The panel of three judges stated that the Trump campaign had not made specific allegations or provided proof according to foreign reports.
The three-judge panel for the US Circuit Court of Appeals denied the Trump campaign's effort to refile the lawsuit, since its allegations had already been rejected by multiple state judges in Pennsylvania.
The 3rd Circuit Court of Appeals had been asked to consider a lower court's decision to dismiss the Trump campaign's attempts to invalidate millions of mail-in votes in Pennsylvania.
The ruling is another major setback for President Trump in his attempts to overturn the 3 November election.
On Thursday he said he would give way if Mr Biden was declared the winner.
The President and some of his allies have been questioning the legitimacy of the election, saying that it was fraudulent and seeking to use legal battles to overturn results in key states.
Biden campaign spokesperson Mike Gwin, following the federal appeals court decision, said that "this election is over and Donald Trump lost" and argued "meritless lawsuits" will not change the outcome.
State and local election officials have said there is no evidence of widespread voter fraud, and both a federal court and the Pennsylvania Supreme Court have dismissed lawsuits seeking to prevent the state from certifying the results of the election.