Bolt had a trial period with Borussia Dortmund last year before starting a similar spell with Australian side Central Coast Mariners in August.
The former sprinter hit a brace in a 4-0 friendly win against Macarthur South West United to prompt suggestions he could be given a deal by the A-League club.
But Bolt's time with the team came to an end in November after he was unable to agree terms on a contract with the Mariners.
Bolt, a Manchester United fan, also rejected the chance to join Maltese club Valletta, while he said he had a number of other offers from teams.
However, Bolt's agent Ricky Simms said on Tuesday that the 32-year-old's budding football career is now over.
"Usain will no longer pursue a career as a professional footballer," Simms said in a short statement provided to Omnisport.
Bolt will go down in history as one of the great athletes of all time, having claimed gold in both the 100metres and 200metres at three consecutive Olympics.