The highly-rated Cheetahs prop was included in coach Jake White's starting XV, after Cobus Visagie suffered an elbow injury during training on Tuesday.
Du Plessis, 24, is the only uncapped player in a much-changed line-up from the one which faced New Zealand, in Durban last month.
Flanker Wikus van Heerden is handed a first Test start since 2003, while fly-half Peter Grant could earn his first cap off the bench.
Du Plessis' younger brother, Bismarck, is also a replacement for Sydney's Telstra Stadium clash.
White has kept just three players who played against the All Blacks in Durban, with Bevin Fortuin preferred at full-back and veteran winger Breyton Paulse returning to the line-up.
There has been much debate surrounding the so-called second stringers that White has brought along, having decided to rest 20 players for the World Cup, and the Springbok coach could not resist teasing the Australians a little more.
"When we lost 49-0 here last year you guys missed the chance of having a go at our B team, you never said a word, or was that our A team that played?" said White.
"But in playing at being selectors for Springbok teams you have been brilliant in motivating my team.
"I must thank you. Before we left South Africa I was not sure what team talk I would give, but now I can just play recordings of what you guys have said and stick press articles on the walls."
White also reminded his opponents that the loss of Lote Tuqiri, who was suspended for breaching team rules, would hurt the Wallabies.
"It is unfortunate for the Wallabies because he is one of the best players in the world. He is a huge loss for the Wallabies. He is a very special player and is very important to their attack. They will miss him on the weekend," said the coach.
"When we do an analysis of the Wallabies not too many teams can afford to lose a player of that calibre."
The Australian Rugby Union (ARU) reported an uptake in ticket sales after the Wallabies win over the All Blacks last weekend.