Lambert was born in Crawfordsville, Indiana. She attended the John Herron School of Art and the Chicago Art Institute to study fashion. Lambart wanted to be a sculptor but went into advertising. She started at an advertising agency in Manhattan, dealing mostly with artists and art galleries.  She was married twice, firstly to Wills Conner, in the 1920s, which ended in divorce and secondly to Seymour Berkson in 1936, which ended with his death in 1959. Eleanor and Seymour had one son together, the renowned poet Bill Berkson. She died in Manhattan in New York City.

In the mid 1930s, Lambert was the first Press Director of the Whitney Museum of American Art and helped with the founding of the Museum of Modern Art. Jackson Pollock, Jacob Epstein, and Isamu Noguchi were a few of the many artists she represented.  In the 1940s, Lambert founded the International Best Dressed List, the Coty Fashion Critics’ Award (in 1943), and New York Fashion Week. In 1959 and 1967, she was asked by the US Department of State to present American fashion for the first time in Russia, Germany, Italy, Australia, Japan, Britain, and Switzerland.  In 1965, she was appointed by President Lyndon Johnson to the National Council on the Arts of the National Endowment for the Arts. In 1962, she organized the Council of Fashion Designers of America (CFDA) and stayed an honorary member until her death in 2003.  In 2001, the CFDA created The Eleanor Lambert Award, that is presented for a “unique contribution to the world of fashion and/or deserves the industry’s special recognition.” Months before she died, she had left her International Best Dressed List to four of Vanity Fair’s editors. Shortly after her last public appearance at New York Fashion Week in September, Lambert died in 2003 at the age of 100. Shortly after her death her grandson, Moses Berkson, completed a documentary film film about her life.