Jones was born Phylis Lee Isley in Tulsa, Oklahoma, the daughter of Flora Mae (née Suber) and Phillip Ross Isley. Her parents toured the Midwest in a traveling tent show they owned and operated. Jones attended Monte Cassino Junior College in Tulsa and Northwestern University, where she was a member of Kappa Alpha Theta sorority before transferring to the American Academy of Dramatic Arts in New York City in 1938. It was here she met and fell in love with fellow acting student Robert Walker. The two were married on January 2, 1939.

They returned to Tulsa for a 13-week radio program arranged by her father, and then headed for Hollywood. Isley landed two small roles, first in a 1939 John Wayne western titled New Frontier, followed by a serial entitled, Dick Tracy's G-Men. In these two films, she was billed as "Phyllis Isley" (Phyllis now spelled with two Ls). However, when she and Walker failed a screen test for Paramount Pictures, they decided to return to New York City.

While Walker found steady work in radio programs, Isley worked part-time modeling hats for the Powers Agency while looking for possible acting jobs. When she learned of auditions for the lead role of Claudia in Rose Franken’s hit play of the same name, she presented herself to David O. Selznick’s New York office but fled in tears after what she thought was a bad reading. Selznick, however, overheard her audition and was impressed enough to have his secretary call her back. Following an interview, she was signed to a seven-year contract.

She was carefully groomed for stardom and given a new name: Jennifer Jones. Director Henry King was impressed by her screen test as Bernadette Soubirous for The Song of Bernadette and she won the coveted role over hundreds of applicants. In 1944, on her 25th birthday, Jones won the Academy Award for Best Actress for her performance as St. Bernadette. That year, Jones' friend, Ingrid Bergman was also a Best Actress nominee for her work in For Whom the Bell Tolls. Jones apologized to Bergman, who replied, "No, Jennifer, your Bernadette was better than my Maria." Jones presented the Best Actress Oscar the following year to Bergman for Gaslight.

Over the next two decades, Jones appeared in a wide range of roles selected by Selznick. Her dark beauty and sensitive nature appealed to audiences and she projected a variable range. Her initial saintly image — as shown in her first starring role — was a stark contrast three years later when she was cast as a provocative bi-racial woman in Selznick’s controversial film Duel in the Sun. Other notable films included Since You Went Away, Love Letters, Cluny Brown, Portrait of Jennie, Madame Bovary, Carrie, Ruby Gentry, Indiscretion of an American Wife, Beat the Devil, Love Is a Many-Splendored Thing, Good Morning Miss Dove, The Man in the Gray Flannel Suit and A Farewell to Arms. Her leading men during this period included Charles Boyer, Joseph Cotten, Gregory Peck, John Garfield, Charlton Heston, Laurence Olivier, Montgomery Clift, Humphrey Bogart, William Holden, Robert Stack, John Gielgud, Rock Hudson, and Jason Robards.

The portrait of Jones for the film Portrait of Jennie was painted by Robert Brackman.

Her last big-screen appearance came in the spectacular disaster film The Towering Inferno (1974), in which she danced with Fred Astairebefore a fire threatened partygoers in a new San Francisco skyscraper.

Jones was a five-time Oscar nominee.

Jones' first marriage produced two sons, Robert Walker Jr. (born April 15, 1940), and Michael Walker (born March 13, 1941). Both later became actors. Jones had a love affair with David O. Selznick, which eventually led to her separation from Walker in November 1943 and divorce in June 1945.

Jones wed Selznick on July 13, 1949, a marriage that lasted until his death on June 22, 1965. After his death, she semi-retired from acting; her last appearance was in 1974's The Towering Inferno. Jones' only child with Selznick, Mary Jennifer Selznick (1954-1976), committed suicide by jumping from a 20th-floor window. This led to Jones' interest in mental health issues.

On May 29, 1971, Jones married multi-millionaire industrialist, art collector and philanthropist Norton Simon, whose son Robert had committed suicide in 1969. Years before, Simon had attempted to buy the portrait of her used in the film Portrait of Jennie. Simon later met Jones at a party hosted by fellow industrialist/art collector Walter Annenberg. Norton Simon died in June 1993. Jennifer Jones-Simon is currently on the board of directors of the Norton Simon Museum in Pasadena.

Jones is a breast cancer survivor. The actress Susan Strasberg, who died of breast cancer, was married to actor Christopher Jones and named her only child Jennifer Robin Jones in the actress's honor.

At age 90, Jennifer Jones enjoys a quiet retirement in Southern California close to her son. She grants no interviews and rarely appears in public.