Wheel 2 Wheel Cycles of Lee on Solent was "highly commended" in last year's running of the award scheme.
Brothers Tony and Graham Childs opened a second outlet last year, in Cowplain.
Co-owner Tony Childs said: "We were stunned to win. We were a bit worried that we werent a sexy enough type of business compared to all the marine and hi-tech companies entering."
The award judges were impressed by the spin-offs the brothers have developed from their original store, such as the Wheel2Wheel travel service, which offers trips for enthusiasts. For example, last year there was an outing to Paris where 30 clients were taken to the finale of the Tour de France.
The shop also supports www.i-team.co.uk, an internet-based cycling club aimed at newcomers to cyclesport.
During Bike Week 2004, Wheel 2 Wheel will be organising BIKEFEST 2004, cycle awareness event for the Portsmouth region.