Semenya crossed the line in a time of 1 minute 58.15 seconds to qualify with the fastest time from the semi-final heats.
Britain's Lynsey Sharp finished second. American Kate Grace - in third - and Bulgaria's Marina Arzamasova joined Semenya and Sharp as the race's four qualifiers.
On Wednesday, Semenya won her opening 800m race in a time of 1:59.31.
She is heavily favoured to win gold in Rio, with commentators touting her "to do something special".
When asked about breaking world records on Wednesday, Semenya said she was more focused on enjoying her championship and running well.
