Cher was awarded the key and plaque on a visit to the Adelaide Grand Prix at the height of her fame in 1990.
Former Lord Mayor Steve Condous, who presented the singer with the prestigious gift, told The Adelaide Advertiser, "I'm disappointed. I would have thought that getting the key to a city like Adelaide would have had some value to her, but obviously it doesn't because she wouldn't have got rid of it."
Current Lord Mayor Stephen Yarwood also said he was "exceptionally disappointed".
"It must be very embarrassing for her if she is selling it because she needs the money," he remarked.
But Cher has responded to backlash from the citizens of Adelaide with a heartfelt shorthand tweet, blaming her "office" for the "fk up".
"I'm upset 2 & trying 2get2 bottom! I Think my office fkd up?" she wrote on Twitter.
Other recipients of the key to Adelaide city include the Dalai Lama, former Governor Dame Roma Mitchell, comedian Barry Humphries and cyclist Lance Armstrong.
According to the eBay listing, the proceeds from the sale of Cher's key will go to charity.