The eldest of three siblings, Moore was born in 1936 in the Brooklyn Heights section of Brooklyn, New York to George Tyler Moore and Marjorie Hackett. She moved to California when she was eight years old.

At the age of 17, Moore started with a role as "Happy Hotpoint" on television commercials broadcast during Ozzie and Harriet. During these commercials she would dance around on the Hotpoint (a General Electric subsidiary) appliances. (Her time as "Happy Hotpoint" ended when her pregnancy, with her only child Richard, became too obvious for her to hide any longer, according to Moore in her autobiography.) She later appeared in several bit parts in movies and on TV shows, including Bourbon Street Beat, 77 Sunset Strip, Surfside Six, Wanted: Dead or Alive, Steve Canyon, Hawaiian Eye and Lock Up in 1961 where a woman named Laura helped save her from prison. Moore's first regular television role was as a telephone receptionist on the show Richard Diamond, Private Detective; in that series, only her legs were shown and voice heard.

In 1961, Carl Reiner cast her in The Dick Van DykeShow, an acclaimed weekly series based on Reiner's own life and career as a writer for Sid Caesar's television variety show, telling the cast from the outset that it would run no more than five years. The show was produced by Danny Thomas's company, and Thomas himself recommended her. Moore's energetic comedic performances as Van Dyke's character's wife, begun at age 24, made both the actress and her signature tight capri pants extremely popular, and she became nationally famous. She won an Emmy award for her portrayal of Laura Petrie. In 1970, after having appeared earlier in a pivotal one-hour musical special called "Dick Van Dykeand the Other Woman", Moore was cast in The Mary Tyler Moore Show, a half-hour newsroom sitcom featuring Ed Asner as her gruff boss Lou Grant, a character that would later be spun off into an hour-long dramatic series.

After a brief respite, Moore threw herself into a completely different genre. She attempted two failed variety series in a row: Mary, which lasted three episodes, and The Mary Tyler Moore Hour, which was canceled within three months. About this time, she also made a one-off musical/variety special for CBS, titled "Mary's Incredible Dream". It did poorly in the ratings. In the mid-1990's, she had a cameo and a guest starring role as herself on two episodes of Ellen. She subsequently also guest starred on Ellen DeGeneres next TV show, The Ellen Show, in 2001. In 2004, Moore reunited with her Dick Van DykeShow castmates for a reunion "episode" called The Dick Van DykeShow Revisited. In August 2005, Moore guest-starred as Christine St. George, a high-strung host of a fictional TV show on three episodes of Fox sitcom That '70s Show. Moore's scenes were shot on the same soundstage where The Mary Tyler Moore Show was filmed in the 1970s.

Moore appeared in several Broadway plays. She appeared in Whose Life Is It Anyway, which opened on Broadway at the Royale Theatre on February 24, 1980 and ran for 96 performances; and Sweet Sue which opened at the Music Box Theatre on Jan. 8, 1988 and ran for 164 performances.

Moore made her film debut in 1961's X-15. She subsequently appeared in a string of 1960s films, including 1967's Thoroughly Modern Millie with Julie Andrews and 1968's What's So Bad About Feeling Good? and Don't Just Stand There!. In 1969 she starred opposite Elvis Presleyas a nun in Change of Habit. Moore was nominated for the Best Actress for 1980's Ordinary People. Other feature film credits include Six Weeks, Just Between Friends, Flirting with Disaster, Labor Pains and Cheats.

In addition to her acting work, Moore is the International Chairman of the Juvenile Diabetes Research Foundation International. In this role, she has used her fame to help raise funds and raise awareness of diabetes mellitus type 1, which she has, almost losing her vision and at least one limb to the disease.