Knight pounced on a mistake by Chesterfield defender Kevin Dawson to fire the decider after 33 minutes.
Dawson fell over as he tried to intercept a pass by Charlie Oatway and Knight gobbled up the opportunity, curling his sixth goal of the season beyond Carl Muggleton.
Chesterfield, now rooted to the foot of the table and still without a win, had been the better team up to that point.
Teenager Ross Flitney, making his debut in goal for Brighton on loan from Fulham, came to the rescue of Steve Coppell's side early on.
The 19-year-old, replacing back injury victim Ben Roberts, did well to save a powerful free kick from Chesterfield captain Steve Payne at the second attempt.
Flitney then almost gifted Chesterfield an equaliser when he dropped a harmless cross by Gus Uhlenbeek. Fortunately for the youngster Oatway was on hand to clear off the line.
Knight nearly doubled Brighton's lead early in the second half. He beat Muggleton to a long through ball by Nathan Jones but stabbed it just wide.
Brighton were nearly made to pay for failing to add to Knight's first half strike. Midfielder Mark Hudson shot narrowly wide for the visitors late on when Flitney lifted clear a Mark Allott corner.
Brighton boss Steve Coppell, paying tribute to Knight, said: "He has got that in his locker. That is what can make him a little bit special. We needed that bit of magic.
He is a good goal scorer, there is no doubt about that. I think teams are trying to stop him from playing now and that is going to be a test of his mental endurance more than anything over the course of the season."
Chesterfield chief Roy McFarland is desperate to add to his squad. "There is a long time to pick up, but we need more experience," he said. "Hopefully we can bring somebody in by Friday."