Tsikhanouskaya was speaking from exile in neighboring Lithuania before an emergency EU summit, due to be held by video conference, to discuss the crisis unfolding in Belarus.
Alexander Lukashenko, the country's veteran leader, has been struggling to contain a wave of mass protests and strikes that pose the biggest challenge to his 26-year hold on power. He denied rigging the August 9 election to secure a sixth term.
Tsikhanouskaya said, speaking in English in a video address, "Mr. Lukashenko has lost all legitimacy in the eyes of our nation and the world."
The opposition politician said she was the rightful winner of the election and wants new elections to be held under some kind of international supervision.
The EU has signaled it will impose sanctions on Belarusian officials it deems responsible for election fraud and a crackdown on protests in which at least two protesters have been killed and thousands detained.