Adrien Silva's application to register for Leicester on a provisional basis was rejected by the Court of Arbitration for Sport.
The midfielder filed his own personal appeal to CAS after the paperwork for his £22 million move from Sporting Lisbon arrived 14 seconds after the summer deadline.
CAS is yet to make a final decision on Silva's appeal, but rejected his request to be registered at Leicester while he waits.
"On 3 October 2017, Adrien Silva filed a statement of appeal at CAS together with the application for urgent provisional measures," a statement read.
"Leicester is not party in this arbitration procedure. Although the player's initial request for interim relief has been rejected, the proceedings will continue and a final decision on the merits will be issued after a hearing, which is yet to be fixed."
An appeal from Leicester and the Football Association to FIFA was rejected in September and Silva is currently unable to play for the Foxes until January.
Silva has already lost his place in the Portugal squad ahead of next year's World Cup and spoke this week about his frustration.
"I had a lot of desire to talk about the injustice," he told Portuguese public broadcaster RTP.
"I was disgusted by this, but people calmed me; they advised me that I had to let go of a painful time to express myself in a calmer, less aggressive way. It was the best decision."
