Winx won her third-consecutive Queen Elizabeth Stakes over 2000 metres, beating Kluger and Hartnell in the 4-million-dollar (2.8 million U.S. dollars) race in front of some 40,000 people at Sydney's sold-out Randwick Racecourse.
Winx will bow out after 43 starts that delivered 37 wins, including 25 at Group One level.
While a win never looked in doubt in Winx's farewell as she strolled to the lead, Japanese-trained Kluger stuck on gamely to finish just 1 1/2 lengths adrift of the world's best racehorse.
She was hot favourite and with long-time jockey Hugh Bowman in the saddle there was only ever going to be one victor, cementing her place in horse racing history alongside fellow Australian greats Phar Lap and Black Caviar. The mighty mare saluted one final time past the post in front of a raucous Royal Randwick crowd.
The win was also her 33rd in a row, with her last loss coming at the corresponding meeting in 2015 when she ran second in the Australian Oaks, a race won on Saturday by stablemate Verry Elleegant who already has the tag of the next champion for Waller.
"It brings the world together as one", trainer Chris Waller said after the win.
The dream team of Bowman and mighty mare Winx.
"This horse has captured the hearts of a lot of people around the globe".
An emotional Bowman said the number of people who had come to see Winx's last-ever race was overwhelming. I mean, she just showed you what she is capable of doing.
Tighe says Kepitis is the most knowledgeable of the trio when it comes to breeding but they will also take advice from others.
"I dare say if she had something else to give, she had more to give".
"In terms of the general sporting public, Winx has gone way beyond being just a figure in horse racing - she is a major sporting figure in the country".
"It sure has. I hadn't thought about winning a race like today ever in my lifetime".
"I can't describe her in words".
Waller has put Winx's imminent retirement to the back of his mind and has been concentrating on preparing his stable super star for her grand final appearance in the Queen Elizabeth Stakes.