Conley, an Irish American, was born south of Chicago in Chicago Heights, Illinois, the daughter of Melba (née Manthey) and Raymond Conley. In the late 1950s and early 1960s, Conley was employed with the traveling theater group the Chicago Uptown Circuit Players and Playwrights Company. She earned a bit part in the movie The Birds, which was followed by similar small roles in movies like Valley of the Dolls and Lady Sings the Blues. Darlene also appeared in the first episode of the fifth season of The Mary Tyler Moore Show as a prison warden.

Darlene played as the tough kitchen supervisor on "Little House on the Prairie" in 1981, in the episode named A Wiser Heart. Her performance as Rose DeVille on The Young and the Restless followed, with Conley playing many small roles on other soaps for most of the 1980s. She was the tough but basically decent prison warden Edith Hopkins on Days of Our Lives in 1983 during Liz Chandler's incarceration for Marie Horton's shooting. As Trixie on General Hospital from 1984 to 1986, she was an old hooker pal of Ruby's who worked at Kelly's. An encore as Rose De Ville inspired william J. Bell to cast her as flamboyant fashion maven Sally Spectra on his newer serial, The Bold and the Beautiful. She earned two Outstanding Supporting Actress Daytime Emmy nominations.

Conley's career spanned fifty years, but she was best known for her performances in daytime television, and in particular, for her portrayal of larger-than-life fashion industrialist Sally Spectra on The Bold and the Beautiful. Conley played the role from 1989 until her death seventeen years later. Darlene's character Sally is the only soap opera character to be displayed at Madame Tussaud's wax figures galleries in Amsterdam and Las Vegas.

Conley became ill in 2004 due to a fall that broke several bones. She recovered from the fall and returned to work (although she was usually seated during her scenes).

The show has kept the role of Sally Spectra alive. Sally's absence has been explained by having her son CJ tell his father, Clarke (and viewers), that Sally had taken a permanent vacation in St. Tropez. In late 2007, Sally decided to sell her fashion house. Nick Marone and Jackie Marone have bought Spectra and are running the company, under the guidance of Clarke Garrison In late January 2017, the show has revisited many of the storylines of Sally Spectra to introduce a new younger character who is supposed to be the niece of Sally Spectra. In addition the show has brought back the original CJ to pay tribute to his mom's office. Despite Darlene Conley's death some 10 years ago, Sally Spectra was shown to be alive and well on the show.