The former Stevenage and AFC Wimbledon front man has signed an 18-month deal with the League One club.
Harrison, 27, joined Cheltenham on a two-and-a-half year deal in January 2013. He scored 15 times last season, striking up an understanding with veteran Jamie Cureton and earning the club's player of the year award.
The Londoner had netted seven times for the Robins this term, including a hat-trick against Swindon Town in the 5-0 FA Cup first round victory.
He made a total of 68 starts and 28 substitute appearances for the club, scoring 23 times.
He fell out of favour with new boss Paul Buckle and was made available for transfer or loan and he has been snapped up by Paul Cook's Spireites, who currently lie ninth in League One following their promotion last season.
Harrison told ctfc.com: "I'd like to just a big thank you to the players and fans, especially after being made supporters' player last season. I really enjoyed my time at Cheltenham and I am looking forward to my new challenge."
"I’ve been very impressed with Chesterfield after having a look around. I like their style of play – it goes well with the type of player I am and I can’t wait to get going."
Buckle said: "On behalf of everyone at the club, I'd like to wish Byron all the best for the future."
Cook told his club's official website: "We have been monitoring Byron closely for some time and we believe he has the necessary attributes to be a valuable addition to our squad."