Templeton was born on December 25, 1865, in Little Rock, Arkansas, where her parents were starring with the Templeton Opera Company. Her father, John Templeton, was a well-known Southern theatre manager, comedian, and author. Her mother, Helen Alice Vane, starred with her husband. At age three, Templeton, dressed as Cupid, sang fairy tale songs between the acts of her father’s plays. Gradually, she was incorporated into the productions as a bit player and then, at five, had actual lines to recite. At eight, she played Puck in A Midsummer Night’s Dream, making her New York debut at Grand Opera House.

At fifteen, Templeton joined a light opera company, playing in a juvenile version of Gilbert and Sullivan’s H.M.S. Pinafore. She also played in The Mascot and Billee Taylor. The same year, she eloped with Billy West, a blackface minstrel performer, but they separated after a honeymoon of six weeks. On October 7, 1885, Templeton had her formal debut in a revival of Evangeline. The play ran for 201 performances. In this show, she displayed talent as both a comedian and mimic. After several years on the road playing in various melodramas and musical farces, Templeton was given the title role in Hendrik Hudson, which opened at the 14th Street Theater on August 18, 1890. It was a “trouser role,” one in which an actress appears in male clothing, then a popular feature of operettas. Her role was of a faithless husband. She won accolades singing “The Same Old Thing,” but the show itself lasted only sixteen performances.

She starred in the London premiere of Monte Cristo Jr., an 1886 hit. Between shows, in 1887, Templeton began living with Howell Osborne, a wealthy broker for Jay Gould; there is no record that they ever married, and the two had no children. The two lived in England and toured the continent for several years. By 1890, Templeton had formed her own opera company and starred in various operettas, none of which fared well financially. In 1895, she starred in another trouser role in E. E. Rice’s Excelsior, Jr. at Oscar Hammerstein’s new Olympic Theater.

In 1896, the comic duo Joe Weber and Lew Fields bought a Broadway theater and formed a stock company made up of headliners, including Templeton. Although she was now buxom even by Gay Nineties standards, her comedic versatility, long dark hair, sultry smile, and throaty-voiced singing won over audiences.

Templeton’s recovered titles are Ma Onliest One, recorded in New York on April 17, 1896, with Len Spencer heard in the vocals, and Rendez Vous Waltz, recorded on July 1, 1897, with music performed by the Metropolitan Orchestra. The gramophone recording process used on these discs was developed by Emile Berliner. The recordings do not contain any evidence of Templeton herself singing or speaking.

In a 1900 show, she introduced John Stromberg’s “Ma Blushin’ Rosie, Ma Posie Sweet,” which became the hit of the show. In 1901, she introduced “I’m a Respectable Working Girl” in the new Music Hall show; she did encore performances several times each evening. After Weber and Fields split, George M. Cohan hired Templeton to play the lead in Forty-Five Minutes from Broadway. Templeton introduced the hit songs “So Long Mary” and “Mary Is a Grand Old Name”. A few months later, she married Pittsburgh industrialist William Patterson, co-founder of Heyl & Patterson Inc., and announced her retirement from the stage; she and Patterson also had no children.

In 1911, Weber and Fields began planning their reunion with a Jubilee touring company featuring all the old Music Hall stars. Templeton was one of the first to volunteer. The tour lasted five months and broke all records for touring companies. She continued in vaudeville with an act that included songs from previous shows. In 1913, Templeton again announced her retirement. Templeton did not make any motion pictures, preferring the live theatre, although she made some phonograph recordings(as songwriter only) and performed on radio shows.

In 1925, she appeared in an old-timer’s show at the Palace Theater, working with Weber and Fields. Asked if she would continue to perform, she replied, “It’s been great fun, but it’s a new Broadway and a new theater, and hereafter I’ll be content to look on from out front”. But she returned to the stage again in 1926 to play Buttercup in a revival of Pinafore. Again, she claimed it was her last appearance on stage. When Templeton’s husband died suddenly in 1932, she returned to the stage to earn a living, appearing in Jerome Kern’s Roberta as Aunt Minnie, a dress shop owner in Paris. Bob Hope, in his American stage debut, handled the comedy. She had only one song, “Yesterdays”. The show ran for nine months.

At age seventy-one and suffering from arthritis, Templeton found herself out of funds and entered the Actors’ Fund Home in New Jersey. On October 3, 1939, at age 73, she died in San Francisco, California, where she had moved to live with a cousin. She was interred in Kensico Cemetery in Valhalla, New York.

In the 1941 musical, Babes on Broadway, Judy Garland performs an impression of Templeton singing Mary’s a Grand Old Name.

In the 1942 movie musical, Yankee Doodle Dandy, Templeton was portrayed by actress Irene Manning.

In the 1968 Broadway musical George M!, she was portrayed by Jacqueline Alloway.

Recordings of music composed by Fay Templeton were preserved on two 1896-97 gramophone recordings discovered in June 2010 aboard the wreck of the Klondike Gold Rush paddlewheeler A. J. Goddard. Other recordings were discovered as well. The Goddard sank in a storm on October 22, 1901 in Lake Laberge, Yukon, and was first found in 2009; the recordings were discovered a year later, after exploration of the vessel.