He finished just ahead of last year's winner, the Etixx Quick Step rider Zdenek Stybar.
"This year I really want to go out with a bang," said Cancellara, 34, who retires at the end of the 2016 season.
"I want to thank all my team-mates for supporting me today, and all these years. This is just amazing!
"I knew that Stybar is a cyclocross guy, so I had to be careful for him. He won last year as well, and he is very good at this last climb. I am very, very happy. Third time…and now they will name a white gravel section after me, and that's exciting."
Gianluca Brambilla finished third after setting the pace for the majority of the race.
He attacked several times in the closing 8km before he was reeled in by Cancellara and Peter Sagan (Tinkoff).
Fabian Cancellara claims third Strade Bianche
"For sure the car gave me the last confidence for the win," said Cancellara after winning the race and the tactical joust.
"They helped me play with the tactics of the Etixx guys. They told me also to let Sagan work (in the chase) and that we don't give too many seconds to Brambilla. He was already in the front all day, so I could catch him in the last climb, and we can play there. Everything went perfectly well.
"I made the pace on the final climb because I knew that Sagan would not do it because I saw him already in a little bit of difficulty. I knew that it was probably going to be Stybar and me, so I had to play like this.
"This is special. I had to play at the end, it was a lottery. I knew I had to risk. I had trust in myself; it was experience. I knew that Etixx was going to play the team game and I just had to make sure that Brambilla did not take too much time.
"The most decisive moment was when Peter made the acceleration and I managed to jump up to him and Stybar. It wasn't a big collaboration between us because we are all big riders, plus Etixx had a team game to play. I think my work in the last 5 kilometers was risky, but it was the only way. It all worked out and I am proud."
Strade Bianche: Siena to Siena, 176km
1. Fabian Cancellara (Swi) Trek-Segafredo in 4-39-35
2. Zdenek Stybar (Cze) Etixx-QuickStep at same time
3. Gianluca Brambilla (Ita) Etixx-QuickStep at 4 secs
4. Peter Sagan (Svk) Tinkoff at 13 secs
5. Petr Vakoc (Cze) Etixx-QuickStep at 34 secs
6. Greg Van Avermaet (Bel) BMC Racing at 37 secs
7. Diego Ulissi (Ita) Lampre-Merida at 41 secs
8. Tiesj Benoot (Bel) Lotto-Soudal
9. Lars Petter Nordhaug (Nor) Sky at same time
10. Alejandro Valverde (Spa) Movistar at 50 secs