The 22-year-old, fresh from his 200m butterfly success on Thursday, finished in 24.24 seconds to take gold ahead of Cameron Brooks-Clarke and Lewis Fraser.
Guy had earlier missed out on the 400m freestyle title, finishing second as City of Sheffield swimmer Jay Lelliott won his first British gold.
Guy said: “There was a lot of hard racing there – I’m happy with how it’s gone.
“I was listening to Craig David when I came out to get me pumped up, he’s been my favourite for a long time,” he added.
Lelliot, who has only recently moved to the City of Sheffield said: “It’s nice to hear British Champion, I’ve never been that before!
“I just wish I could have gone quicker.”
Elsewhere on Friday, Siobhan-Marie O’Connor won gold in the women’s 200m individual medley and Yorkshire’s Lizzie Simmonds – the Beverley-born swimmer – claimed the 200m backstroke title.
O’Connor said: “I’m really pleased with the swims I have done here and delighted to have won with some amazing girls.
“It’s been a really good week but now I’m looking forward to going and making my final preparations for the Commonwealth Games,” she added.
Scotland’s Lucy Hope took the 50m freestyle crown while Holly Hibbott and Chloe Tutton were crowned British champions in the 100m breaststroke and 800m freestyle respectively.
In the men’s events, James Wilby won the 200m breaststroke ahead of Commonwealth champion Ross Murdoch while Chris Walker-Hebborn claimed the 100m backstroke title.
Wilby said: ““It was a bIt of a surprise to be honest.
“I was practising a few new things so was pretty happy with how it went.”