If Geoffrey and Dawn Bonelle are successful it could open up the floodgates to hundreds of similar challenges.
The couple from Livingston, West Lothian, claim their original £30 fine - imposed after Geoffrey, 67, drove through a bus gate in October 2014 - was unfair due to a lack warning signs.
Motorists have been hit with nearly £3 million in penalty charges for driving through the gate at Nelson Mandela Place, in Glasgow city centre, in just over a year.
The couple eventually paid a £274 fine because they were worried the council might try to make a claim on their possessions, including their home.
But now they are launching a civil case to recoup that cost and hope it may become a test case that will allow others to claim their fines are unfair.
Dawn, 59, said: “We want our day in court to see if the sheriff can make the council prove these signs were not only legal but were in the correct place and correct height.
“They have made a lot of money from this bus gate. We hope this case would open the doors for thousands of other drivers.
“We want to claim the money back because we feel they can’t prove and haven’t proved the signs were not only legal, but put on the right place, at the right height.”
Unlike speed camera fines, which go the UK Treasury, fines from bus lane cameras are retained by the city council for transport improvements.
Greg Whyte, of legal firm Jones Whyte Law, said if the court found in their favour it would open up avenues for others to make legal challenges.
He said: “The crux of their case is that area is not properly signposted.
“If a sheriff at Glasgow Sheriff Court rules that they are correct, it opens the floodgates for those who have one of these tickets to say they didn’t see it either and had no reason to believe it was a bus lane.”
Motorists caught by the camera at Nelson Mandela Place paid out £1.3m in fines after 70,000 charge notices were issued to car drivers between the end of June 2014 and the end of July 2015.
The council say 44,000 fines have been paid to date and many of them had been reduced from £60 to £30 because they were settled within two weeks.
The council then warned outstanding fines would be pursued, and passed to a debt recovery agency if necessary. If all are paid, that would produce another £1.5 million.
Bus lane fines paid across the city totalled £1.6 million in 2014 and £1.2 million in the first half of 2015 compared to nearly £3.3 million in 2013.
A Glasgow City Council spokesman said: “Numerous signs and variable messaging have been in place to alert drivers to the bus gate at Nelson Mandela Place since June 2014.
“Since then the number of fixed penalty notices issued on this location has dropped significantly as people are choosing not to drive through what is now a very well-known and publicised bus gate.”