Comic Enterprises successfully argued in the High Court that 20th Century Fox's show breached its trademark right to The Glee Club name.
The show's broadcaster appealed against the 2014 ruling, but appeal court judges dismissed their case.
It is not yet clear whether the Glee show's name will have to be changed.
The appeal court judges are now due to consider whether EU trademark law affects the ruling and therefore means no change is required.
More updates on this and Birmingham and the Black Country
Part of the film studio's argument was that it should not need to change the name, citing EU law which states a trademark must be "a sign", in the sense of being a single sign and capable of being "graphically represented".  The show's name, it said, did not fall into this category.
As a result, the judges have asked the two parties and the UK Intellectual Property Office to enter their submissions on the issue by Monday when they will consider whether to send the case to the European courts.
The television show first aired in 2009 on the Fox Channel - part of Rupert Murdoch's media empire.
Mark Tughan, owner of the comedy club chain, said he received an 80-page ruling from Lord Justice Lloyd Jones, Lady Justice Arden and Lord Justice Kitchin on Monday following the appeal hearing in November.
Mr Tughan said the comedy club's trademark was registered in 1999 and that he started legal proceedings in 2011.
The film studio is yet to comment on the appeal being thrown out.
Mr Tughan, who tweeted his victory, told BBC News "I feel vindicated, not only for taking the case in the first place but now that two courts have come to the same conclusion about the infringement on my trademark.
"One would hope that Fox would put down their weapons but they have shown no inkling of wanting to settle this."
He added he was awarded some compensation from the High Court hearing but said he is yet to pursue what he feels he is entitled to.
The Glee Club opened in Birmingham in 1994 and now has branches in Cardiff, Nottingham and Oxford.

The man, who is 22 years old, is charged with procuring the drugs for his co-defendant, a 21-year-old woman.
The woman is charged with taking two drugs which are commonly used to bring about a miscarriage or abortion.
At Belfast magistrates court on Monday, there was an application to adjourn the case.
The man's solicitor said there had had been a lot of unacceptable delay in the case and that it was quite stressful for his client.
The case was adjourned until 23 January.
In Northern Ireland, unlike the rest of the UK, abortion is only allowed if a woman's life is at risk or if there is a permanent or serious risk to her physical or mental health.
The makers of hit TV show Glee have lost their appeal against a legal ruling that found in favour of a comedy club chain of the same name.
[NEXT_CONCEPT]
A solicitor for a man accused of procuring drugs intended to cause an abortion has protested about delays in dealing with the case.