Boles was born in Greenville, Texas to a middle-class family. He graduated with honors from the University of Texas in 1917. He returned to Greenville, where he was selected by an out-of-town producer to act in an opera at the King Opera House. This experience convinced John that he preferred music and the stage to the preference of his parents, who wanted him to pursue a medical degree. He married Marcelite Dobbs in that same year. His parents wanted him to become a physician. He studied and got his B.A. but never went to medical school due to the allure of the stage. During World War I, he was a US spy in Germany, Bulgaria, and Turkey. He started out in Hollywood in silent movies, but became a huge star with the advent of talkies. After the war, Boles moved to New York to study music. He quickly became well known for his talents and was selected to replace the leading man in the 1923 Broadway musical Little Jesse James. He became an established star on Broadway and attracted the attention of Hollywood producers and actors. He was hired by MGM to appear in a silent film in 1924. He starred in two more films for that studio before returning to New York and the stage. In 1927, he returned to Hollywood to star in The Love of Sunya (1927) opposite Gloria Swanson, which was a huge success for him. Unfortunately, because the movies were still silent he was unable to show off his singing ability until late in the decade. In 1929, Warner Brothers hired him to star in their lavish musical operetta The Desert Song (1929). This film featured sequences in Technicolor and was a box-office success. Soon after, Radio Pictures (later known as RKO) selected him to play the leading man in their extravagant production (the last portion of the film was photographed in Technicolor) of Rio Rita, opposite Bebe Daniels. Audiences were enthralled by his beautiful voice, and John Boles suddenly found himself in huge demand. RCA Victor even hired him to make phonograph records of songs that he had sung in his films.

As soon as Rio Rita was completed, Boles went back to Warner Brothers as the leading man in an even more extravagant musical entitled Song of the West (1930) that was filmed entirely in Technicolor. Shortly after this film, Universal Pictures offered John Boles a contract, which he accepted. He starred in a number of pictures for them, most notably the all-Technicolor musical revue entitled The King of Jazz (1930) and a historical operetta entitled Captain of the Guard (1930). In 1931, he starred in One Heavenly Night (1931), which would prove to be his last major musical. Boles portrayed Victor Moritz in the original Universal version of Frankenstein (1931). He starred with Irene Dunne in a 1934 film adaptation of Edith Wharton’s 1920 novel The Age of Innocence directed for RKO Radio Pictures by Philip Moeller, and took the role of Edward Morgan in Curly Top (1935), starring Shirley Temple. In 1937, Boles starred alongside Barbara Stanwyck in the King Vidor classic Stella Dallas. In 1943, he co-starred with Mary Martin and Kenny Baker in One Touch of Venus. Boles retired from the screen and stage in 1952. He died in 1969, aged 73. He was survived by his wife and two daughters.