The Inter Milan winger, on loan from Sassuolo, struck in the fourth minute of added time after stepping off the bench in the 87th minute.
Italy failed to qualify for the Nations League finals at the weekend after a 0-0 stalemate against Portugal and Roberto Mancini's new-look side appeared to have drawn another blank.
But Politano broke the deadlock with the last kick of the game to condemn the USA to their fifth defeat of the year.
Federico Chiesa twice threatened for Italy in the opening exchanges. His angled shot was well saved by Ethan Horvath and another effort from outside the penalty area flew narrowly wide.
Domenico Berardi's header soon after was just off target and Leonardo Bonucci's shot was well saved by Horvath as Italy chased the opening goal.
The Americans' starting XI had an average age of 22 years and 71 days, but they stuck doggedly to their task.
Berardi skewed an ambitious shot from out wide way off target and the Sassuolo forward forced Horvath into another fine save with a shot from outside the box.
Horvath rescued his side again just before the interval when he palmed away Marco Verratti's goal-bound free-kick as the first half ended goalless.
Mancini handed a senior debut to Hoffenheim midfielder Vincenzo Grifo, who replaced Federico Chiesa at the break.
Juventus teenager Moise Kean won his first senior cap when he replaced Berardi just after the hour before USA threatened for the first time in the match.
Kellyn Acosta provided the cross and Walker Zimmerman's header was turned over the crossbar by Italy goalkeeper Salvatore Sirigu.
Italy cranked up the pressure. Horvath produced another excellent save to keep out Grifo's low shot, Francesco Acerbi fired wide following a corner and Stefano Sensi went close on his debut.
USA almost snatched a 78th-minute lead through Bobby Wood after Tyler Adams' cushioned header as Italy committed more men forward.
Kevin Lasagna made a hash of his shot from in front of goal and in the 87th minute was replaced by Politano, who won his side the match.
Fellow substitute Roberto Gagliardini picked him out in the box and Politano took the ball in his stride before burying the last kick of the match into the bottom corner.