The three-time world champion, a silver medallist at the Tokyo Games three years ago, clocked 12.99 seconds for victory.
Teammate Daniel Roberts claimed silver in 13.09sec in a photo-finish with Jamaica's Rasheed Broadbell.
Olympic gold was the one medal missing in Holloway's impressive haul.
The 26-year-old American is the second-fastest man in history in the event, with a personal best of 12.81sec.
He was surprisingly beaten into silver at the Covid-delayed Tokyo Games in 2021 by Jamaican Hansle Parchment but has been untouchable since then.
Parchment finished eighth and last in the final in 13.39sec.
