The 25-year-old Jamaican retained his title and won the gold medal with an Olympic record time of 9.63 seconds.
Bolt's race, which also featured Americans Justin Gatlin and Tyson Gay and fellow Jamaican star Yohan Blake, drew 19.4 million viewers to BBC One last night.
Meanwhile, 628k watched the race on the BBC's Olympics 3 channel.
The Jamaican sprinter will be back in action on Tuesday (August 7) in the 200m heats. The 200m final takes place on Thursday (August 9) at 8.55pm.
BBC One's average audience for last night's Olympics coverage, which included Christine Ohuruogu securing a silver for Team GB in the women's 400m final, was 12.9m (a 50.9% share).
[London 2012 - In Pictures]
Copyright: Martin Rickett/PA Wire/Press Association Images