The 58-year-old funnyman and presenter, who has twice suffered heart attacks, will be playing cancer patient Tim Connor in three episodes of the popular show which begin screening in September.
Paul - also a Radio 2 host - said he was delighted to be appearing and had long been an admirer of the programme.
He said: "I'm a big fan of Holby City so to be featuring in three episodes is absolutely brilliant."
The TV star is also to return to early evening chat shows on ITV later this year and won acclaim for his series For The Love Of Dogs. He is also narrating an ITV documentary Me And My Guide Dog on July 3.
Holby City executive producer Oliver Kent said: "Having Paul appear in Holby City is such a great coup for the show.
"Paul has a vibrant personality and a very unique way of engaging with the audience - not to mention he's incredibly funny. This is exactly what we need for a storyline that will be filled with a rollercoaster of emotions."