The 35-year-old has already won two golds and was hoping to add a third medal in the women’s pair with team-mate Polly Swann of Scotland
But the duo finished fourth, meaning Glover has come up short in her bid to be the first woman to win at three different Games.
"In Rio I said it was my last one – this time I’m saying, ‘no, that’s definitely it’,” she said afterwards.
There was further agony for Team GB as Emily Craig and Imogen Grant missed out on a medal place by 0.01 seconds in a dramatic lightweight women’s double sculls event.
Britain has now finished fourth in five events at the Sea Forest Waterway complex and has just one silver medal claimed by the men’s quadruple sculls team on Wednesday, despite topping the medals charts in rowing at Beijing 2008, London 2012, and Rio 2016, claiming 20 medals in total.
Glover added: "We're pleased with the result; it shows our hard work."
The mother-of-three, who gave birth to twins Kit and Willow in 2020, is the first mum to turn out for GB’s rowing team and began her comeback journey last March with the suspension of the Tokyo Games due to the coronavirus pandemic allowed her a third shot at the Olympics.
She linked up again with Swann – who took her own break from rowing to focus on her medical career – after Heather Stanning took time off after London 2012.
"This has felt more like a journey than anything we've done,” Glover explained. “We even look to the route of getting to the start line and how many crews fall to the wayside.
"You can never say that a place in the final isn't exciting.
"This last year for both of us is one we're going to look back on and I think when you're caught up in the moment of it and the day-to-day grind of only having one year, it feels so immediate.
"I'll come to look back in a few years and think, 'how did I do that?' 'What was that year about?'"
Swann, an NHS doctor, was also seeking to cap a stunning comeback after taking a break to focus on her medical career.
The former George Heriot’s pupil began her rowing career on Edinburgh’s Union Canal as a 14-year-old and in the early stages of the pandemic she worked as a junior doctor at St John’s Hospital in Livingston.
"For Helen, she was looking after three kids. For me, I was working in a hospital a year ago today," Swann said afterwards.
"I don't think there are many people in the Olympic athlete set-up who can say these things and be in a final.
"We gave our all to try and get onto that podium. I can't fault our determination for that, or the way that we fought every day in training."
Team GB are back in rowing action in tomorrow’s men’s eight and women's single sculls.