Stapleton was born in Manhattan, New York City, to Joseph E. Murray (a billboard advertising salesman) and Marie Stapleton Murray (an opera singer). At age 18, while she was a student at Manhattan’s Hunter College, she began her career in 1941 in summer stock theatre, and made her New York debut in the Off-Broadway play American Gothic.  Stapleton was featured on Broadway in several hit musicals, such as Damn Yankees, Funny Girl, Bells Are Ringing, and Juno. She guest starred in many television series, including the role of Rosa Criley in the 1963 episode “The Bride Wore Pink” on NBC’s medical drama about psychiatry, The Eleventh Hour.  Stapleton’s early work on television included roles in Starlight Theatre, Robert Montgomery Presents, Lux Video Theater, Woman with a Past, The Philco-Goodyear Television Playhouse, Dr. Kildare, My Three Sons, Car 54, Where Are You?, Dennis the Menace, and Naked City. On the episode “The Hidden Jungle” of The Defenders broadcast on December 1, 1962, Jean Stapleton guest-starred as Mrs. Larsen with future television husband Carroll O’Connor.

In 1979, Stapleton appeared in the original Canadian production of the musical Something’s Afoot, which was later broadcast on Showtime. In 1982, Stapleton portrayed Eleanor Roosevelt in a television production of the First Lady’s later life. In 1998, and for a few years afterward, Stapleton took her “Eleanor” characterization to live theaters, now adapted as a one-woman show. She co-starred with Whoopi Goldberg in Bagdad Cafe, the television series based on the movie of the same name and made a guest appearance September 30, 1978 on the sixth episode of the third season of The Muppet Show.

Stapleton’s awards for All in the Family include three Emmys and two Golden Globes. She was offered a role in the feature film Willy Wonka & the Chocolate Factory as Mrs. Teevee, but she declined because it coincided with the production of the All in the Family pilot (the role went to Nora Denney).  Stapleton appeared in made-for-TV movies and feature films such as Klute, the comedy Cold Turkey (a 1971 Norman Lear film) and the Faerie Tale Theatre episode “Cinderella” as the fairy godmother and as the Giant’s Wife in “Jack and the Beanstalk”. She also had a recurring role on television’s Scarecrow and Mrs. King as a British spy. She appeared in the educational series Beakman’s World as Beakman’s mother, Beakmom. Stapleton co-starred in 1984’s feature film The Buddy System with Susan Sarandon and Richard Dreyfus.

She declined the opportunity to lead in the television mystery programme Murder, She Wrote, which from 1984 to 1996 instead starred Angela Lansbury.  In 1996, Stapleton played opposite John Travolta, portraying the eccentric rooming house owner, Pansy Milbank in Nora Ephron’s hit Michael. Stapleton also appeared in the 1998 feature You’ve Got Mail as a close co-worker in whom Meg Ryan’s character confides. On May 14, 2000 in the “Mother’s Day” episode of the TV series Touched by an Angel she played an angel named Emma who came to help Celine who was taking care of her late best friend’s mother.

In the 1990s, Stapleton played the role of Mrs. Piggle-Wiggle in a children’s series of the same name based on the books by Betty MacDonald.  In 1996, Stapleton appeared on Everybody Loves Raymond playing Ray’s imperious aunt. That same year, she also appeared in the Murphy Brown episode “All in the Family” playing Miles’s grandmother, Nana Silverberg. Making a debut in the world of video games, Stapleton was the voice of Grandma Ollie on KinderActive, Turner Pictures, and New Line Cinema’s venture Grandma Ollie’s Morphabet Soup. The game won a Teacher’s Choice Award from Learning Magazine. On 26 January 1998, Stapleton guest-starred on the Jean Smart sitcom, Style & Substance in the episode “A Recipe for Disaster”, playing a former television chef who has an alcohol problem.  Stapleton voiced John Rolfe’s maid, Mrs. Jenkins, in Disney’s 1998 direct-to-video animated film Pocahontas II: Journey to a New World.

Stapleton rarely appeared on talk shows, but she reunited on-camera with O’Connor on Donny and Marie Osmond’s show on April 24, 2000, a little over a year before O’Connor’s death. When the show’s hosts asked her to perform in Edith Bunker’s voice, she jokingly told them that she only does so “for pay”.  Stapleton seldom watched All In the Family, but by 2000, she reconsidered, she said to the Archive of American Television.  A 2001 made-for-TV movie, Like Mother, Like Son: The Strange Story of Sante and Kenny Kimes, starred Mary Tyler Moore as Sante Kimes, Gabriel Olds as Kenny, and Jean Stapleton as Silverman.  In 2002, Jean Stapleton was inducted into the American Theatre Hall of Fame. She was also inducted into the Television Hall of Fame that same year.

For 30 years, Stapleton’s husband William Putch (1924–83) directed a summer stock theater, Totem Pole Playhouse, at Caledonia State Park in Fayetteville, Pennsylvania. Stapleton performed regularly at the theatre with the resident company. Together they had two children: actor/writer/director John Putch and television producer Pamela Putch.  Stapleton’s brother, Jack Stapleton, was a stage actor. Her cousin is actress Betty Jane Watson. Stapleton was active in the Christian Science church.  Jean was not, as commonly believed, related patrilineally to the actress Maureen Stapleton. Genealogists have determined that the two were fourth cousins through Jean’s maternal grandmother and Maureen’s paternal grandmother.

Stapleton died, surrounded by family and friends, on May 31, 2013, in New York City, of natural causes. She was 90 and is survived by her two children, John, a TV director, and Pamela, a TV producer.  Norman Lear said, “No one gave more profound ‘how to be a human being’ lessons than Jean Stapleton.” Fellow US sitcom actress Roseanne Barr said that Stapleton’s range was “unbelievable, deep and majestic.” Co-star and BAFTA- and Oscar-nominated director and producer Rob Reiner said, “Working with her was one of the greatest experiences of my life.” Sally Struthers said, “Jean lived so in the present. She was a Christian Scientist who didn’t say or think a negative thing … She was just a walking, living angel”.  The marquee lights on Broadway were dimmed for one minute on June 5, 2013 at 8 p.m. EDT to honor the memory of Stapleton.