Semenya finished behind American Alysia Johnson Montano in a time of 2:00.71, after Montano clocked in at 2:00.44.
Halima Hachlaf of Morocco took the final qualification spot, finishing third in 2:00.99.
Semenya will now compete in the 800m semifinal on Thursday at 8.30pm (SA time) ahead of the final on Saturday evening, where she will need to vastly improve on her qualification time in the heats, as she faces stiff competition heading into the semifinal.
Favourite Pamela Jelimo from Kenya has on five separate occasions beaten Semenya's best time of the season, 1:59.18.
It's been nearly three years since her astonishing victory in the 2009 Berlin World Championships, which turned from a dream win into a nightmare, as she was forced to undergo a series of drug and gender tests after that victory and subsequent performances were questioned by officials, athletes and an often unsympathetic public.
She was prevented from competing for almost a year before being cleared for competition, only to have her comeback hampered by a niggling back injury.
The 21-year-old is now being coached by Maria Mutola of Mozambique, the 800m champion who won gold at the 2000 Sydney Olympics, and is confident ahead of competition, and is not interested in dwelling on the past.
"You need to concentrate on the future. I don't have to entertain those [setbacks] now. I can concentrate on my running, that's all," Semenya told AFP recently.
"That's what I have to learn. I have to act professionally. But this is my first Olympics so it'll be special for me."