The 19-year-old was starting at the heart of La Roja team alongside Manchester City enforcer Rodri and Paris Saint-Germain's Fabian Ruiz.
Despite having already secured their place in the top two of Group A alongside Scotland - meaning the 2012 winners had sealed their spot at next summer's tournament - Luis de la Fuente named a strong XI, including the likes of Jose Gaya and Dani Carvajal.
One of only two players to retain their spots from the victory at Cyprus earlier in the international break, centre-back Robin Le Normand put the hosts ahead in the fourth minute.
Napoli attacker Khvicha Kvaratskhelia restored parity for the visiting Georgians in the 10th minute, and not long after Gavi found himself in a great deal of pain after attempting to control the ball.
The Barcelona youngster was in clear discomfort and although he was able to walk off the pitch with some assistance from Spain's physio, it was obvious that the midfielder was in no state to play any further part.
Barca teammate Ferran Torres put La Roja back into the lead early in the second half and celebrated by holding up a Gavi shirt, suggesting that his compatriot were already aware of how serious the injury is set to be.
An own goal from Luka Lochoshvili of Serie B side Cremonese made sure of all three points for Spain, with De la Fuente's men finishing four points clear of Steve Clarke's Scotland at the top of Group A.
Since the final whistle in Valladolid, it has been confirmed that Gavi has suffered a serious anterior cruciate ligament injury, meaning he is set for a significant period on the sidelines.
It is understood that the 19-year-old will now return to Barca for detailed examinations on Monday that will help determine the specific timeline for recovery.
Gavi has been a regular figure for the Catalonians since making his debut in July 2021, appearing on 111 occasions across all competitions and netting seven times.
So far this season Barcelona head coach Xavi has fielded the youngster in 12 La Liga matches - 10 of which from the start - with the midfielder bagging one goal and one assist.
Due to the long-term injuries sustained by fellow engine room options Frenkie de Jong and Pedri earlier in the campaign, Gavi has found himself as a mainstay in the Spanish champions' XI.
Barca currently sit behind perennial challengers Real Madrid and surprise package Girona in the La Liga standings after 13 matches, and Xavi's side will be looking to close the gap to first when they travel to Rayo Vallecano on Saturday afternoon.