The Briton, chasing his third win in a row at the Circuit de Catalunya and fourth in total, lapped with a best time of one minute 16.568 on an overcast morning.
Ferrari's Charles Leclerc was 0.531 slower, with championship leader Valtteri Bottas third and 0.555 off Hamilton's pace after going off and into the gravel.
Sebastian Vettel, Leclerc's team mate, was fourth on the timesheets in 1:17.172.
Hamilton is a point behind Bottas, who was quickest in both Friday sessions, after four successive one-two finishes for Mercedes -- the best ever start to a Formula One season by any team.
News that makes sense
Your trusted source for staying up-to-date with the world around you. Get free daily news updates and analysis, straight to your inbox.
With two wins each, the Finn is ahead by virtue of a point for fastest lap in the Australian season-opener.
The U.S.-owned Haas team continued their strong showing in practice with Frenchman Romain Grosjean fifth and team mate Kevin Magnussen sixth.
Red Bull's Max Verstappen, who in 2016 became the youngest Formula One winner at 18 years old at the Spanish circuit, was seventh for Red Bull.
British rookie George Russell spun his Williams off and backwards against the tyre wall in the closing seconds.
(Reporting by Alan Baldwin, editing by Christian Radnedge)
