A run of four birdies from the 13th to the 16th in the afternoon on the Old Course had put the three-time major winner five under and in position to enter a six-man play-off for the final four spots available.
But he recorded a bogey five to miss out as South Africans Garth Mulroy, Thomas Aiken, Tjaart van der Walt and Australian Marcus Fraser qualified from the extra holes with Welshman Oliver Farr missing out.
“It was disappointing to bogey the last,” Harrington said. “I nearly walked off after four holes this morning so I was surprised to have a chance at all.
“It was nice to get a run going on the back nine. It’s one too many, so it is disappointing, especially after making a few birdies and getting inside the mark.”
Two-time European Tour winner Alexander Levy will make his US Open debut at Chambers Bay next month after finishing top of the leaderboard at 11 under following a 66 on the Old Course and a 67 in the afternoon.
“I’m really happy. I played really good golf for the two 18 holes,” he said. “I was positive and had a good attitude.
“It’s nice to play the US Open. I was a little upset to slip out of the top 60 before Wentworth so it’s great to now get in.
“It will be my first time in the US Open. I missed the Masters, so it will be great to play in another major.”
Englishmen John Parry and Jason Palmer also qualified with the latter likely to get attention in the United States due to his unusual one-handed chipping style.
“The chipping is incredibly unique but it has saved my career, because there is no way I’d be competing as a professional if I didn’t find the one-handed method,” he said.
“At the end of last year I went from 50th on the Challenge Tour to having a win, a second place, and being on The European Tour and now this. I’m having an incredible time. I love the game of golf and at the moment it is being good to me.
“I doubt there have been many one handed chippers at the US Open before. The rough might be a bit too deep to play one-handed, so I’ll have to assess it when I get there, but it is a problem I’m looking forward to have.”
India’s Shiv Kapur, Swede Alex Noren, Denmark’s Lucas Bjerregaard and German Marcel Siem were the other qualifiers with Shane Lowry, Matteo Manassero and Nicolas Colsaerts among those to miss out.
Meanwhile, Wen-Chong Liang, Masahiro Kawamura, Hiroyuki Fujita, Kurt Barnes and Baek Seuk-hyun all progressed from the qualifying tournament in Japan.