Five members took Labour to court over their right to vote after the party ruled that only those who were members before 12 January this year.
Labour's ruling committee proposed that new members would only be able to vote if they paid £25 to become "registered supporters" in a 48 hour window.
And a court ruled in favour of the members earlier this week - meaning that up to 130,000 people had joined the party since January would now be able to vote.
However, the party has today won out in the court of appeal, which said that Labour's national executive committee was the "ultimate arbiter" of party rules.
The judgement said: "A member’s entitlement to vote in a leadership election is not a product of him or her simply being a member, but is the result of him or her being a member who satisfies the precise eligibility criteria defined by the NEC and any freeze date provisions set by the NEC in the timetable for the election."
The five Labour members have been denied permission to take the case to the supreme court.
Labour challenger Owen Smith had speculated that many of the 130,000 members would back incumbent leader Jeremy Corbyn, and his campaign team had called for the leadership battle to be extended by up to two weeks.
Labour NEC chair Paddy Lillis said: "It was right that the Party appealed the judgement on the freeze date, just as we would have appealed if the Court in the previous case did not uphold the NEC decision that the incumbent leader of the Labour Party did not require nominations.
"It is crucial to the Labour Party that our governing body has the authority to debate, decide and implement the procedures, timetable and voting eligibility for our internal elections and selections."
However, Corbyn responded that the verdict was "the wrong decision".
"The Court's ruling disenfranchises nearly 130,000 Labour members, who joined the party since January and were explicitly told that they would have a vote in any leadership election," he said.
"Serious questions must be raised, however, over why and how the NEC Procedures Committee brought this appeal. In doing so, it effectively risked new members’ money on an attempt to disenfranchise them.
"If we are to build a big, inclusive party to take on the Tories, we need to secure democracy in our party."