Prince William was in the audience, although he and Middleton weren't yet an item.
Some commentators maintain that the black see-through dress with a blue trim played a role in sparking the royal romance that started shortly after the event. It was initially intended to be worn as a skirt, but Middleton wore it as a short dress with black lingerie underneath.
It was designed by Charlotte Todd, who now works at an aquarium.
The dress was bought by an unnamed buyer at Kerry Taylor Auctions for 65,000 pounds ($104,948) and an additional 13,000 pounds ($20,989) of buyer's premium.
"He thinks it's an iconic piece," said an unnamed man who represented the buyer at the auction. "He's very happy."
About three to four bidders competed for the dress in the packed room, while some also bid by phone. Gasps rang around the room when the bidding drove the price up to 65,000 pounds ($104,948).
William and Middleton are set to marry at Westminister Abbey in London on April 29.
The Associated Press contributed to this report.