The Tasmanian posted a time of nine minutes and 10 seconds in the 7km stage against the clock around Porto-Vecchio.
Manuele Boaro (Saxo-Tinkoff) and Tejay van Garderen (BMC) were a second further back with Porte's British team-mate Chris Froome fourth, a further second back.
The course was the same as last year when Cadel Evans beat Michael Rogers by a fraction of a second but the 2011 Tour de France champion and defending champion Evans could only post a time of 9:56 this year.
Porte will hold the overall lead going into Sunday's final stage which ends with a 14km climb of the Col de L’Ospedale. But the Paris-Nice champion will be heavily challenged with a cluster of top riders within striking distance.
"It was quite a technical route this afternoon, and not my sort of a course," said Porte after the win.
"But I've done a lot of work on my time trial position, and that's definitely paid dividends.
"We're in a good position with me in the lead and Froomey sitting just a few seconds back. It's going to be quite tight tomorrow because there's a lot of good riders close to us.
"We came in with Froomey as our leader and he's a great friend, so if he's got good legs tomorrow and I don't then I'll be more than happy to ride for him. Obviously, I'd like to finish the job off if I can, but as long as Team Sky win tomorrow it'll be a great day and we'll all be happy.
"Right now I just want to enjoy the moment, and the fact that I have one more lion to add to the collection (laughs). I'm really happy on this team and in a really good place.
"There won't be much celebrating tonight because we're all focused on tomorrow. We've got a great team here to defend and that's what we'll aim to do."