The Spaniard, who prevailed on the Vuelta in 2008 and 2012, controlled his rivals in the final ascent and struck with just over one kilometre left to counter Britain's Chris Froome.
The Team Sky rider had set the pace in the climb to Puerto de Ancares to defend his second place overall and he was almost empty when Contador attacked, finishing about 16 seconds behind according to provisional results.
Spain's Alejandro Valverde retained third place overall by taking third place at the end of the 185.7-km ride from Santo Estevo de Ribas de Sil.
The top three are unlikely to change after Sunday's final stage, a 9.7-km individual time trial around Santiago de Compostella.
At the base of the 12.7-km final ascent - at an average gradient of 8.7 per cent - Pole Przemyslaw Niemiec of Poland was on his own with a 30-second advantage over the peloton.
He was quickly swallowed up and Joaquim Rodriguez, fourth overall and hoping for a podium finish on Sunday, attacked with just under 9 km left.
His podium spot threatened, Valverde went to try and bridge the gap with a trio of Froome, Contador and Italian Fabio Aru just behind him.
Also feeling Rodriguez's threat, Froome had to work to control the Spaniard's lead while Contador was happy to just stay on his wheel.
But Froome's acceleration hurt everyone and Valverde was dropped.
Froome and Contador reeled in Rodriguez 5.5 km from the line with 'Purito' cracking about one kilometre further up and being passed by Valverde.
Tinkoff-Saxo rider Contador leads Froome by one minute 37 seconds overall and Valverde (Movistar) by 2:35.
Both Contador and Froome are taking part in the Vuelta after crashing out separately from the Tour de France last July.
(Writing by Julien Pretot; Editing by Alan Baldwin)
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