Laugh in the Face of Cancer 5 at City Varieties Music Hall takes place on Wednesday.
The line-up includes compere Matt Reed, Lucy Beaumont, Stephen Cookson and Jarred Christmas - and a mystery guest is also due to appear.
Elizabeth and Jordan Long have organised the event annually for five years.
Family of tragic Birstall teenager ask public not to donate to Go Fund PageIt has raised £34,000 for Leeds Cares in honour of Mrs Long's sister Alex Beaumont, who was diagnosed with breast cancer.
Although she needed a double mastectomy and had to have her lymph nodes removed, Mr Long said she is getting better.
With colleagues at their workplace, Lowell, the Rothwell organisers have helped £140,000 over recent years.
Mr Long, 32, said that he and his wife decided to start putting on the show after going to a stand-up event in Chapel Allerton.
"We realised we could do this - we could put on a bigger show," he said.
"At the end of the day, our theory is that laughter is the best medicine."
The likes of Joe Lycett have previously appeared at the event.
Mr Long said: "The comedians will do it for free and they are fantastic in helping support the event itself."
Tickets for the show, which begins at 7.30pm, cost £15.
For more information, visit www.cityvarieties.co.uk or call the box office.