His special cameo marked his first major TV appearance since he took part in the We Are Manchester concert in 2017.
Ahead of the charity telethon, the comedian, 46, asked members of the public to film themselves walking along to the Tony Christie track in a nod to his 2005 Comic Relief stunt.
The original clip saw Kay and celebrity pals lipsyncing along to the song while marching through a series of weird and wonderful landscapes.
In a video broadcast during Thursday's charity telethon, the comedian said that he “never imaged” the song would go down so well when it was first made, adding that it is the “perfect time” to recreate it.
The video featured families at home, shop workers, firemen and women and health care workers.
Thank you @comicrelief @BBCCiN and of course @peterkay_co_uk for putting together such a wonderful new video to ‘that song’! Hope the nation enjoyed, was a pleasure to be a part of. #BigNightInAmarillo #BigNightIn #StayAtHome pic.twitter.com/gxYADwSR2k
— Tony Christie (@TheTonyChristie)
In the clip the footage of people dancing to the music is interspersed with clips of Kay from the original 2005 video.
The comedian appeared on the programme via a video which was filmed in his garden.
He said: “I never imagined that it would go down so well and make everybody happy, and I thought what a perfect time to recreate it again, so I’ve asked a lot of you to film yourself and send it in.”
The comedian ended his message saying “take care and enjoy yourselves, lots of love to you all”.
With additional reporting by Press Association