Horne was born in Bradford, Pennsylvania. Her parents moved to Long Beach, California, near Los Angeles, when she was 11. She studied voice at the University of Southern California and participated in Lotte Lehmann's vocal classes.

Horne's first major professional engagement was in 1954, when she dubbed the voice of Dorothy Dandridge in the film Carmen Jones. Up to that point, she had worked as a background singer for several TV sitcoms, as well as recorded covers of popular songs of the early 50s. She made her Los Angeles debut the same year when she performed the role of Hata in The Bartered Bride with the Los Angeles Guild Opera.

Her first major breakthrough came when her singing ability was recognized by Igor Stravinsky; her operatic career began when he invited her to perform in the 1956 Vienna festival. She remained in Europe for three seasons singing for Oper Gelsenkirchen. She was highly acclaimed for her performance as Marie in Berg's Wozzeck at the inauguration of Gelsenkirchen's new opera house on May 22, 1960.

In 1964, she returned to the United States to appear in Wozzeck at the San Francisco Opera.

For many years, Horne was associated with the Australian soprano Joan Sutherland. They first performed together in a concert version of Vincenzo Bellini's Beatrice di Tenda at Carnegie Hall in February, 1961. In 1965, they were paired again in a performance of Rossini's Semiramide with the Opera Company of Boston.

Horne made her debut at the Royal Opera House, Covent Garden in October 1964 as Marie in Wozzeck. Her La Scala debut was as Jocasta in Stravinsky's Oedipus Rex on March 13, 1969. Another one of Horne's breakthroughs occurred in 1969 during a performance of Rossini's Le Siège de Corinthe at La Scala, when Horne received a remarkable mid-act seven minute ovation. Horne made her debut at the Metropolitan Opera in 1970 as Adalgisa in Bellini's Norma with Joan Sutherland in the title role. She thereafter appeared regularly at the Met, opening the 1972-1973 season as Carmen. In 1984, she sang the title role of Handel's opera seria Rinaldo, the first Handel opera ever performed at the Met.

Although best known for her bel canto and opera seria roles, Horne also performed much American music, both contemporary music, by composers such as William Bolcom, and traditional popular songs.

Horne was married from 1960 to 1979 to the conductor Henry Lewis.

Horne retired from the concert stage in 1999 with a recital at the Chicago Symphony Center. She still occasionally performs at pop concerts (most recently with cabaret star Barbara Cook), her voice undimmed by age. Horne has also established the Marilyn Horne Foundation to help fund music education.

Many music critics consider Horne to be one of the greatest singers in the history of opera. She is seen as the prototypical American singer; brisk, efficient, earnest, technically secure and no "diva" traits, perhaps only missing a touch of the irony and darkness of spirit of old world singers.

It was reported in January 2006, shortly after Horne's 72nd birthday, that she had been diagnosed the previous month with localized pancreatic cancer, and that her prognosis is good.