The Supreme Court of Justice described the appeal as “inadmissible”.
After the ruling Henrique Capriles tweeted that there was a “lack of justice” in Venezuela.
Nicolas Maduro, Hugo Chavez’s handpicked successor, won the election by less than 1.5 percentage points – about 200,000 votes.
The opposition alleged that the vote had been marred by fraud.
In June the Venezuelan National Electoral Commission (CNE) confirmed President Nicolas Maduro’s victory in an audit on millions of votes, but Henrique Capriles denounced the audit as “a fake”.
In her ruling on Wednesday, Justice Gladys Gutierrez said the opposition had not offered “sufficient proof” to back up their allegations.
The 10-point appeal had been lodged by Henrique Capriles, the opposition coalition and several citizens.
On Tuesday, Henrique Capriles criticized the court’s delay in making a ruling and said that he would take the case to “international bodies”.
April’s election was called after the death of Hugo Chavez on March 5 following a long battle against cancer.
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