Tymoshenko, surrounded by a media scrum, appeared at the supreme administrative court in person to file a complaint demanding that the declaration of the final results be quashed.
She and her supporters carried several boxes which a statement on her website said contained evidence, including video recordings and photographs, that the February 7 election was fraudulent.
"We are sure that there were systemic, fundamental and general falsifications of the elections in the second round," Tymoshenko said as she filed the complaint, the Interfax news agency reported.
Yanukovych defeated Tymoshenko by a narrow margin of around 3.5 percent or just under 890,000 votes in the election, according to complete official results published on Sunday.
International observers have described the elections as fair and democratic and Western governments have joined Russia in congratulating Yanukovych on his victory.
Tymoshenko has said observers from the transatlantic security group the OSCE were willing to testify in court about vote fraud, which she alleges amounted to one million votes.
However the head of the OSCE short-term election observers during the election, Joao Soares, Monday denied his group had any information or evidence of any fraud in the election.
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