Substitute Isco struck deep in stoppage time to earn Spain a 2-2 draw and deprive Gareth Southgate of victory in his final game as England interim manager on Tuesday.
Goals from Adam Lallana and Jamie Vardy had set England up for an impressive win, but Spain debutant Iago Aspas reduced the arrears in the 89th minute before Iscolevelled with practically the last kick.
Southgate, who lost Lallana to injury in the first half, cursed his luck on the touchline, but having gone four games unbeaten since replacing Sam Allardyce, the job is still expected to be his full-time.
"The players over the period of the four matches couldn't have given any more," Southgate said.
"It feels like a defeat for the players, but I couldn't be more proud of what they've done. It was a healthy, positive performance."
While victory eluded England at the last, the vibrant nature of their performance at Wembley will help to erase lingering memories of their Euro 2016 humiliation by Iceland.
Isco's 96th-minute strike extended Julen Lopetegui's unbeaten record to six games since he succeeded Vicente del Bosque as Spain coach in the aftermath of their Euro 2016 exit at Italy's hands.
"In the first half England were better than us. They started really sharply," said Lopetegui.
"Things got even tougher in the second half, but that's when we showed our character and never-say-die spirit.
"To pull a draw out of the fire with two goals at the end was great. It showed we never give up."
Vardy won the penalty that yielded England's opening goal in the ninth minute, but he was perhaps fortunate to still be on the pitch.
With less than five minutes on the clock, the Leicester City star followed through after clearing the ball downfield and caught Cesar Azpilicueta high on the Chelsea defender's right shin.
Vardy does the Mannequin
But Romanian referee Ovidiu Alin Hategan kept his cards in his pocket and within four minutes he was pointing to Spain's penalty spot.
After Jesse Lingard robbed Thiago Alcantara, Lallana curved a gorgeous pass into the box from the right and despite a loose first touch, Vardy succeeded in drawing a foul from goalkeeper Pepe Reina.
Reina went the right way from the penalty, to his left, but Lallana put enough height on the ball to register his third goal in three England outings.
The fates were to conspire against the Liverpool midfielder midway through the first half, however, as injury forced him off, with Theo Walcott coming on in his place.
Shy of a mishit Vitolo shot, Spain had failed to trouble England and Lopetegui changed tack on the half-hour, ditching his initial 3-5-1-1 system for a more conventional 4-1-4-1.
But the traffic remained one-way in the second half and after Walcott had seen a shot deflected into the side-netting, England made it 2-0 in the 48th minute.
Stand-in captain Jordan Henderson's cross from the right was met with a brilliant diving header from Vardy, who had previously gone 14 games without scoring for club and country.
He celebrated by freezing stockstill in the manner of the 'Mannequin Challenge' social media fad, with Walcott and Sterling joining in.
Lopetegui awarded senior debuts to Ander Herrera and Aspas in the second half, yet still the hosts advanced, Reina saving one-handed from Walcott and Nacho clearing off the line from Lingard.
When Spain did threaten, England substitute goalkeeper Tom Heaton denied David Silva and substitute Alvaro Morara, with Aspas putting the rebound over the bar in the first instance.
West Ham United left-back Aaron Cresswell won his first England cap as a late substitute, but was upstaged by Celta Vigo forward Aspas and Real Madrid midfielder Isco, his fellow entrants.
Aspas halved the deficit, drifting in from the right and curling home, before Iscoatoned for an earlier miss by chesting down Dani Carvajal's pass and squeezing a shot between Heaton's legs.