Moreno was born Rosa Dolores Alverío in Humacao, Puerto Rico, to Rosa María, a seamstress, and Paco Alverío, a farmer. She moved with her mother to New York City at the age of five, and took the surname of her stepfather, Edward Moreno. She began her first dancing lessons soon after arriving in the United States from a friend of her mother, a Spanish dancer called Paco Cansino who was the uncle of Rita Hayworth. When she was eleven years old, she lent her voice to Spanish-language versions of American films.

She had her first Broadway role—as "Angelina" in Skydrift --by the time she was 13, which caught the attention of Hollywood talent scouts. She appeared in small roles in The Toast of New Orleans and Singin' in the Rain, in which she played Zelda Zanners, a fictional silent screen vamp. In 1956, she had a supporting role in the film version of The King and I as Tuptim, but disliked most of her other work during this period.

In 1961, Moreno landed the role of Anita in Robert Wise's film adaptation of Leonard Bernstein's and Stephen Sondheim's groundbreaking Broadway musical, West Side Story, which was played by Chita Rivera on Broadway. Moreno won the Best Supporting Actress Academy Award for that role. Moreno went on to be the first actress (and the first Hispanic) to win an Emmy (1977), a Grammy (1972), an Oscar (1962) and a Tony (1975). In 1985, she won the Sarah Siddons Award for her work in Chicago.

Besides appearing in Singin' in the Rain, The King and I, Summer and Smoke (1961), West Side Story, The Night of the Following Day (1968) and Carnal Knowledge in (1971), Moreno appeared on the PBS children's series The Electric Company in the 1970s, most notably as Millie the Helper. In fact, it was Moreno who screamed the show's opening line, "HEY, YOU GUYS!" She also had roles as the naughty little girl Pandora, and as "Otto", the very short-tempered director. Moreno appeared in the family variety series The Muppet Show, and she made other guest appearances on television series such as The Rockford Files, The Love Boat, The Cosby Show, George Lopez, The Golden Girls, and Miami Vice. She was also a regular on the short-lived sitcom version of Nine to Five (based on the film hit) during the early 1980s.

Moreno's Broadway credits include The Last of the Red Hot Lovers, Gantry, The Ritz, for which she won the 1975 Tony Award for Best Featured Actress, and the female version of The Odd Couple. In 1993 she was invited to perform at President Bill Clinton's inauguration and later that month was asked to perform at the White House. During the mid 1990s, Moreno provided the voice of Carmen Sandiego on the animated Fox show Where on Earth is Carmen Sandiego?

In the late 1990s, she gained exposure to a new generation of viewers when she played Sister Pete, a nun trained as a psychologist in the popular HBO series, Oz. She made a guest appearance on The Nanny as Coach Stone, Maggie's (Nicholle Tom) tyrannical gym teacher, whom Fran Fine (Fran Drescher) also remembered from her school as Ms. Wickavich.

Moreno continues to be active on stage and screen. In 2006, she portrayed Amanda Wingfield in Berkeley Repertory Theatre's revival of The Glass Menagerie. She was seen on Law and Order: Criminal Intent as the dying mother of Detective Goren. She was a regular on the short-lived TV series Cane, which starred Jimmy Smits and Hector Elizondo.

On June 18, 1965, Moreno married Leonard Gordon, he was her husband and manager, until he died on June 30, 2010. They have one daughter, Fernanda Luisa Gordon-Fisher, and two grandsons, Justin and Cameron Fisher.

Among Moreno's awards and recognitions are the following:

Rita Moreno (1961) · Patty Duke (1962) · Margaret Rutherford (1963) · Lila Kedrova (1964) · Shelley Winters (1965) · Sandy Dennis (1966) · Estelle Parsons (1967) · Ruth Gordon (1968) · Goldie Hawn (1969) · Helen Hayes (1970) · Cloris Leachman (1971) · Eileen Heckart (1972) · Tatum O'Neal (1973) · Ingrid Bergman (1974) · Lee Grant (1975) · Beatrice Straight (1976) · Vanessa Redgrave (1977) · Maggie Smith (1978) · Meryl Streep (1979) · Mary Steenburgen (1980)