He effectively closed any chance for a new vote by calling the election an "absolute victory," the Associated Press reported.
Supporters of candidate Mir Hossein Mousavi have been protesting election results all week by marching in Tehran streets, conjuring memories of the 1979 Islamic Revolution, with the next major demonstration planned for Saturday afternoon.
Speaking at Friday prayers at Tehran University, Khamenei also accused foreign media and Western countries of trying to create a political rift and stir up chaos in Iran, according to the AP.
"Some of our enemies in different parts of the world intended to depict this absolute victory, this definitive victory, as a doubtful victory," he said. "It is your victory. They cannot manipulate it."
The supreme leader said the Obama administration has passed along mixed messages about relations between the United States and Iran, saying the president lauded the street protests but also extends an olive branch to the government, CNN reported.
"Which one should we believe?" he asked.
Khamenei said the 11 million votes that separated Ahmadinejad from his top opponent, Mousavi, were proof that fraud did not occur.
"If the difference was 100,000 or 500,000 or 1 million, well, one may say fraud could have happened. But how can one rig 11 million votes?" Khamenei asked.
He reiterated that he has ordered the Guardian Council, an unelected body of 12 clerics and Islamic law experts, to investigate the other candidates' voter fraud claims.
Mousavi has called for annulment of the election result, which showed Ahmadinejad the winner with nearly 63 percent of the vote to 34 percent for his closest challenger, Reuters reported.
After Khamenei spoke, another defeated candidate, Mehdi Karoubi, also called for the result to be nullified.
Meanwhile, the U.S. House of Representatives approved a resolution by a 405-1 vote Friday condemning Tehran's crackdown on demonstrators and the government's interference with Internet and cell phone communications.
Rep. Mike Pence, R-Ind., who co-sponsored the resolution, said he disagrees with the Obama administration's stance that it must not meddle in Iran's affairs.
Rep. Ron Paul, a Texas libertarian, cast the sole opposing vote because he said it wasn't the House's place to judge "events thousands of miles away about which we know very little."
With the ongoing unrest in Iran and the government's crackdown on foreign journalists and certain Web sites, Google launched an online Persian (Farsi) text translation service Friday at http://translate.google.com to help people understand the information trickling out of Iran as news stories, Facebook messages, blogs, e-mail and other form of communication.
Social media sites like Facebook and Twitter have been cited as key organizing tools for the political protesters.