Gloria Reuben was born in Toronto, and was raised there and in nearby London, Ontario, as the second youngest of six children. She credits the early influence of her mother, an accomplished singer, and her older brother, a Broadway actor, for inspiring her to develop her performing talents. She began playing piano as a child and studied ballet, jazz, and music technique and theory at the prestigious Canadian Royal Conservatory.

While Reuben enjoyed a brief modeling stint in Canada, she appeared in commercials and in small roles on Canadian television. In 1988, she relocated to Los Angeles and raised her television profile as a recurring regular on "The Round Table" and as a guest star on "The Young Riders" and "China Beach" (a series produced by "ER" executive producer John Wells), among other series. Soon after, Reuben appeared as a homicide detective in three episodes of NBC's "Homicide: Life on the Street" and she began on "ER" in a recurring role as Jeanie Boulet, a married therapist who provided home care for Dr. Benton's (Eriq La Salle) ailing mother and found herself falling in love with Benton. When she joined "ER" as a series regular in the second season, her character was promoted to a physician assistant and soon ended her rocky romance with Benton.