The ruling on Thursday cleared the way for the central election commission to publish the final, official results of the 26 December repeat vote and officially declare "orange revolution" leader Viktor Yushchenko the winner.
"The court has decided to reject the appeal," chief presiding justice Andriy Hnatenko said.
"The court decision is final and cannot be appealed."
In his appeal, Yanukovich has asked the court to overturn a 30 December decision by the central election commission, which refused to grant his request and review the election results on the grounds that it was riddled with fraud.
Viktor Yanukovich has contested
The commission said at the time that there was a lack of evidence of fraud. But Yanukovich charged that the commission made its ruling before it had received all relevant information.
The three-justice panel did not agree.
"The court could not conclude that the central election commission was negligent and did not find lawful basis to strike down its decision," Hnatenko said.
Yushchenko won the 26 December election with 52% of the vote compared with Yanukovich's 41%, a difference of more than 2.2 million ballots on a turnout of 77%.
The historic rematch between Yushchenko and Yanukovich was set after an earlier November election was annulled by the Supreme Court because of massive fraud.
The election was annulled amid massive protests organised by the opposition, which saw hundreds of thousands of people clad in the orange colour of Yushchenko's campaign bring Kiev to a virtual standstill.