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Mr Trump alleged, without evidence, that President-elect Joe Biden's votes in Pennsylvania were created "out of thin air" in an early morning tweet on Saturday.
A panel of three judges on Friday voted unanimously to toss out the case, saying that team Trump had provided no evidence of voter fraud.
"Charges require specific allegations and then proof. We have neither here," judge Stephanos Bibas wrote in the court's ruling.
Team Trump's case suggested poll watchers were banned from observing the vote-counting process. They also argued the state illegally allowed counties to decide whether voters could fix mail-in ballots with missing signatures or secrecy envelopes.
The defeat is the latest set back for Mr Trump, 74, and his lawyers' attempts to defy the will of the American people by overturning an election in which he lost both the electoral college and popular vote.
Undeterred by the Pennsylvania defeat, Mr Trump launched into another midnight hour Twitter tirade, repeating his baseless claims of ballot irregularity.
"I won Pennsylvania by a lot, perhaps more than anyone will ever know," said the defeated incumbent. "The Pennsylvania votes were RIGGED. All other swing states also. The world is watching!"
Mr Trump lost Pennsylvania by some 81,660 votes, according to the Associated Press's most up-to-date count.
And As judge Bibas alluded to, there is so far no evidence of voter fraud in the state, despite team Trump's claims.
President-elect Joe Biden secured 3,459,923 votes in Pennsylvania, turning the Rust State blue after it went red for Trump in 2016.
The 3rd Circuit Court of Appeals had been asked to consider team Trump's case after it had been thrown out by a lower court.
The lower court's decision to dismiss the case laid the ground for election chiefs in Pennsylvania to officially declare Mr Biden the winner in the state, picking up its 20 electoral college votes.
Electoral college officials are scheduled to meet on 14 December, a date that some Trump insiders have admitted could be his last stand.
In a scathing 21-page ruling which appeared to directly attack Mr Trump's attempts to subvert democracy in the state, judge Bibas said “free, fair elections are the lifeblood of our democracy”.
He added: "Charges of unfairness are serious. But calling an election unfair does not make it so."
Judge Bibas also pointed out that the number of ballots team Trump targeted in its challenge was far smaller than his margin of defeat and therefore inconsequential to the result.
Rudy Giuliani, who is Mr Trump's personal lawyer and leading his election results challenges, said he would take the fight to the Supreme Court.
“The activist judicial machinery in Pennsylvania continues to cover up the allegations of massive fraud,” he and fellow campaign attorney, Jenna Ellis, said in a statement following the ruling.
Trump has also challenged the results in Michigan, Nevada, Arizona and Georgia but suffered multiple defeats, with most legal experts in agreement that the lawsuits are destined to fail.