Born Anna McKim in New York, New York, Dvorak was the daughter of silent actress Anna Lehr and the actor/director, Samuel McKim, and as a child appeared in several films.

She began working for MGM in the late 1920s as a dance instructor and gradually began to appear on film in small musical roles. Howard Hughes groomed her as a dramatic actress and she was a success in such pre-Code films as Scarface (1932), as Paul Muni's sister; as the doomed unstable "Vivian" in Three on a Match (1932), co-starring Joan Blondell (who died the same month that Dvorak did in December, 1979), and Bette Davis; and opposite Spencer Tracy in Sky Devils (1932).

Known for her style and elegance, she was a popular leading for Warner Brothers during the 1930s, and appeared in numerous contemporary romances and melodramas. A dispute over her pay (she discovered she was making the same amount of money as the little boy who played her son in Three on a Match) led to her finishing out her contract on permanent suspension, and then working as a freelancer, but although she worked regularly, the quality of her scripts declined sharply.

With her British husband, the actor Leslie Fenton, Dvorak travelled to England where she supported the war effort by working as an ambulance driver, and worked in several British films. She retired from the screen in 1951, when she married her 3rd and final husband (Nicholas Wade), to whom she remained married until his death in 1977. She had no children.

She lived her post-retirement years in anonymity until her death (from undisclosed causes) in Honolulu, Hawaii at the age of 68. She was cremated and her ashes scattered.

Ann Dvorak has a star on the Hollywood Walk of Fame for her contribution to Motion Pictures, at 6321 Hollywood Boulevard.