Williams has been stuck on 23 victories, one behind Margaret Court, since January 2017 and lost in remarkable circumstances to Naomi Osaka in last year's New York final, as well as falling at the final hurdle at Wimbledon in each of the past two seasons.
It had appeared as though the 37-year-old would finally end her wait for the landmark triumph with an outstanding run back at Flushing Meadows until she met giant-slayer Andreescu on Sunday (AEST).
The teenager, who was beaten in qualifying last year, has never lost to a top-10 player and continued that incredible record by triumphing 6-3 7-5, becoming the first Canadian to win a singles grand slam.
Williams wore a pained expression throughout a performance that became more and more ragged, gifting away three breaks to double-faults as her experience failed to stand up to a mature Andreescu display.
For 23 minutes at the end of the second set, it appeared there might be a miraculous fightback, as the pressure finally got to the previously indomitable Andreescu, who had led 5-1 in the second.
But her calm and composure was rediscovered in the nick of time and the major final debutant, who was not born when Williams won the 1999 title, sealed a stunning triumph, leaving Serena still level with Chris Evert for US Open championships (six) and match wins (101).