Retour
The Lions – who play in the Northern Premier League Premier Division – had made a bright start with Elliot Owen and Oliver Brown both going close before midfielder Jack Emmett saw his header cleared off the line just before the break.
There was a delay to the start of the second half because of a medical incident in the crowd, with the match restarting around 15 minutes late.
Sutton improved in the second half, with defender Ben Brookes firing across the face of goal and Temi Babalola heading over from a corner.
Guiseley, though, secured a memorable cup win when Thewlis slotted in from the spot in the fourth minute of added time.