"It's so stupid I'm speechless," he said. "I've never had batteries on my bike."
A video featuring former professional cyclist Davide Cassani seemingly showed a bike operating with what he claimed was a motor concealed by the pedals, with a button to operate the device hidden on the handlebars.
In Cassani's video, there is footage of Cancellara and what it claims are suspicious slips of his right hand on the bike handlebars before he acclerates effortlessly away from his rivals.
"It's quite funny but it's become a bigger story and is no longer so funny," said Cancellara. "It's a sad and really outrageous story. Believe me, my feats are the result of hard work."
The sport's governing body, the UCI, confirmed that there was no case against the Swiss rider.
And the UCI's technical chief, Jean Wauthier, told Belgian daily newspaper Het Laatste Nieuws that it was unlikely Cancellara would use any kind of motor.
"The risk is simply too big. For him, his team and the bike manufacturers. A champion like Cancellara would not take that risk."