Isner came back from a set down to beat world No. 5 Zverev 6-7 (4/7), 6-4, 6-4 in a clash which lasted for two hours and 29 minutes.
En route to his first ever Masters 1,000 title, The 14th seed defeated in-form Juan Martin del Potro in the semi-finals and put behind his three previous major tour final.
The 32-year-old had lost his previous three major tour final appearances in 2012, 2013 and 2016.
"To win like that in front of a crowd like that, with that atmosphere, you can't replicate moments like that. It was absolutely amazing," said Isner post his title triumph.
"This tournament has so much history. All the best players have played here through the years. For Sascha and I to share the court in the last men's singles match ever here at this tournament is amazing. I never thought I would be at this moment considering how I was playing coming into this event."
With this win, Isner has become the first US player since Andy Roddick in 2010 to lift the Miami title. Also, the victory will take him to move up by eight spots to match his career-high of No. 9. (ANI)