Hollywood star and licensed commercial pilot, John Travolta will fly them over for the eight-day trip.
Winfrey will also film an episode of her show at the Sydney “Oprah” House.
The Australian government said it was spending AUS$3 million (£1.8m) to help bring The Oprah Winfrey Show to the country as a way to boost tourism.
Tourism Minister Martin Ferguson insisted it was money well spent as it would put Australia in the spotlight, with the popular TV show watched by 40 million Americans and screened in 145 countries.
“Oprah is a household name and her star power has the potential to lift Australia’s profile as a premier tourist destination,” he said in a statement.
Winfrey made the announcement during the first episode of her 25th and final series on Monday.
After teasing the audience with a suggestion she might take them on a trip to New York, Philadelphia or Los Angeles, she said her last season merited something “bigger”.
“I started to think about where would I most want to go. Maybe I should take all of you with me to the other side of the world. We’re going to Australia!” she said.
Several episodes will be filmed during the trip, which Winfrey said had been almost a year in the planning and would take in beaches, vineyards, Sydney Harbour and the Great Barrier Reef.
It marks the first time that the show will be filmed with its US audience outside North America.
The Australian trip follows a similar stunt in 2004 when Winfrey gave a car to each member of her audience.
The 56-year-old is due to launch her own television channel, the Oprah Winfrey Network, in 2011.