Drivers will be hit with a £60 fine if they use bus lanes in Lancaster from Tuesday (July 16).
Cameras have been monitoring the bus lanes on Morecambe Road and Parliament Street since the start of June.
Another bus lane over the Greyhound Bridge is currently closed while the bridge is being repainted, however, this lane will also be enforced as soon as the work is finished.
Until now anyone driving in the bus lane has received a warning letter instead of a penalty notice to give people a chance to get used to them being enforced.
But from 6am on July 16, anyone using the bus lanes will receive a fine. The standard Penalty Charge Notice (PCN) is £60, which is reduced to £30 if paid within 14 days. The bus lanes will be enforced 24 hours a day, 7 days a week. They can also be used by cyclists.
The introduction of the new bus lane on the Greyhound Bridge last year means that buses will no longer have to merge into general traffic when leaving the bus stop on Cable Street, instead simply continuing along the bus lane.
At the end of the bus lane, general traffic in the right hand lane is directed to merge in with buses so that, when traffic is queued up, buses have priority and can progress further along the queue.
County Coun Keith Iddon, cabinet member for highways and transport, said: "The bus lanes are there to give priority for buses, and enforcing them helps to ensure that everyone follows the rules, and the system works as it should.
"I hope that we don't make a penny from penalty notices, as it will mean that people aren't abusing the bus lanes. There is very prominent signage, which means everyone should know not to drive in the bus lanes."
The work to repaint the Greyhound Bridge is underway with the bus lane on the bridge expected to be back in use around the end of September 2019.