Born Juanita Emylyn Pique in Fulton, Kentucky, she grew up in St. Louis, Missouri. In 1936, she told a Harvard Crimson interviewer, "I guess I'm just a natural dancer". She recalled performing professionally albeit underage at age 11 in a "Kids Act". She was seen and hired by vaudevillian Gus Edwards and taken on tour; at one stop, "child labor authorities hauled her ... off the stage".
She continued to work in vaudeville and on stage. Mayfair was in at least four Broadway productions in the 1930s, including the last edition of Flo Ziegfeld's Follies in 1931. She joined the cast of At Home Abroad when star Eleanor Powell, also discovered by Gus Edwards, had to leave the show.
According to the Pittsburgh Post-Gazette, "the manager of the Main Street Theater in Kansas City" did not like her name, and changed it to Mitzi Mayfair without her knowledge; when she first saw the name on the marquee, she thought she had been replaced. However, the Brooklyn Daily Eagle had a different story, stating that Gus Edwards forgot her name and made one up.
During World War II, Mayfair embarked on a USO tour of Europe and North Africa with the likes of Kay Francis, Carole Landis and Martha Raye. All four performers played themselves in the film recreation of the tour, Four Jills in a Jeep (1944). Mayfair appeared in a number of shorts, but this and Paramount on Parade (1930) were her only feature film credits. The celebrated dancer Irene Castle considered having Mayfair (among others) play her in the film The Story of Vernon and Irene Castle, but decided she was not a big enough star. As Fred Astaire was already cast as Vernon, the part went to Ginger Rogers.
She was first married to Albert F. Hoffman from 1938 to 1943. On April 7, 1944, she married Charles Henderson, "associate boss of the music department of the 20th Century-Fox Studio". It is unclear when this marriage ended. On June 28, 1963, Mayfair married Fred S. Cook of Kitsap County, Washington. She died in Pima, Arizona in May 1976 at age 61.