Tymoshenko said that more than one million votes which would have handed her victory had been subject to fraud.
According to preliminary official figures, Yanukovich beat her by 3.5 percentage points with about 880,000 more votes.
Tymoshenko´s stubborn refusal to concede to Yanukovich flies in the face of the West´s endorsement of the official result. On Friday, U.S. President Barack Obama led world leaders, the European Union and NATO in congratulating him on his victory.
Yanukovich, a 59-year-old ex-mechanic from the Donbass mining region, is expected to tilt Ukraine back towards Russia, after five years of estrangement under pro-Western President Viktor Yushchenko.
The Tymoshenko camp says Western leaders have backed him because they are fearful of unrest breaking out in the ex-Soviet state of 46 million.
But she said some OSCE observers had proof, including video material, of "systematic falsification" in the voting and were ready to speak out on this in court.
Her continued refusal to concede victory to Yanukovich or heed his call to resign as prime minister spells continuing turmoil for Ukraine.
Tymoshenko´s camp is then expected to challenge this in a Kiev high court on the basis of the "proof" which it says it has compiled from hundreds of polling stations around the country.
But many analysts doubt that there is sufficient proof to persuade the court this time to overturn the commission´s findings.