Ayuso, who went solo to win stage seven, had Romo for company for the final 25 kilometres of the 144.9km ride from Laredo to Los Corrales de Buelna and timed his final move to perfection to overtake Romo close to the line.
"After such a hard day, you don't know, and I had to play my cards," Ayuso said, adding that his team car had instructed him to ride tactically and make Romo believe he would need to "pull more" if he wanted a chance of winning the stage.
"I had to make him a bit nervous," he said.
France's Brieuc Rolland (Groupama-FDJ) came in 13 seconds behind in third having threatened to bridge the gap in the final kilometres, while Vingegaard (Visma-Lease a Bike) finished safely in the peloton which ambled in over six minutes later.
There was no change at the top of the general classification, with Vingegaard maintaining his 50-second lead over Portugal's Joao Almeida (UAE Team Emirates-XRG) with Briton Tom Pidcock (Q36.5 Pro Cycling Team) a further six seconds back in third.
Ayuso has had an eventful Vuelta so far. Aside from his two stage wins, having begun as one of the riders tipped to challenge favourite Vingegaard, the 22-year-old has also lost chunks of time after getting dropped on climbs.
The Spaniard's exit from UAE Team Emirates-XRG was announced on Monday's rest day, much to the anger of Ayuso who heavily criticised the timing of the statement the following day.
At the halfway stage, a breakaway group of over 50 riders were over three minutes ahead of the peloton, and as the leading pack was whittled down, Ayuso tried to get away on his own on the final climb.
Romo went with him, and with Rolland still chasing, the leading pair began their game of cat and mouse in the final kilometre, and it was Ayuso who came from behind on the last turn before the line to outsprint Romo.
The runner-up was left punching his handlebars in frustration, coming so close to a first Grand Tour stage win.
"It's not something I really enjoy, not cooperating fully, but sometimes you have to play smart," Ayuso said.
"And that's what I did in the final and the sprint."
After the drama of the previous day in the Basque Country, where pro-Palestinian protesters brought a premature end to the stage with no winner, it was a more relaxed stage in Cantabria.
The GC leaders appeared to be holding their fire for Friday's stage 13 which will be one of the toughest of this year's race.
A 202.7km mountain stage awaits, with all the climbing coming in the latter part where the riders tackle three category one climbs including the brutal ascent to the finish at Angliru, where the Vuelta could be won and lost.