Ukraine unrealistically needed to better Slovakia's result in Luxembourg to earn an automatic berth at the tournament in France next year but their fellow Eastern Europeans secured a 4-2 win to make sure of second place.
With Spain already assured of top spot, coach Vicente del Bosque fielded an experimental team at Kiev's Olympic Stadium, scene of La Roja's 4-0 triumph against Italy in the final of Euro 2012.
Fullback Mario Gaspar scored with a header on his debut to put Spain ahead in the 22nd minute and Cesc Fabregas, captaining the side on his 100th international appearance, won a penalty moments later but had his effort saved by Ukraine goalkeeper Andriy Pyatov.
Spain's eighth qualifying win in a row put them on 27 points from 10 matches and they have gone eight competitive matches without conceding for the first time. Slovakia have 22 points and Ukraine 19, eight ahead of fourth-placed Belarus.
Ukraine had a number of chances in the early stages and David De Gea, deputising for captain Iker Casillas in goal, had to make two brilliant saves to deny Artem Kravets and Ruslan Rotan after 15 minutes.
Spain made the breakthrough when Nolito sent Thiago Alcantara clear and his perfect cross to the far post was headed powerfully into the net by Gaspar with Pyatov stranded.
Nolito played Fabregas in just after the restart and he was felled by Oleksandr Kucher but Pyatov flung out an arm to send the midfielder's spot kick over the bar.
Fabregas, the 10th player to reach 100 caps for La Roja and the second-youngest after Sergio Ramos, has missed all four of his penalties for Spain excluding shootouts.
Ukraine went close to an equaliser when Andriy Yarmolenko struck a shot against the frame of the goal in the 51st minute before Paco Alcacer was denied by another fine Pyatov save.
Substitute Serhiy Rybalka had a great chance to level a minute from time but De Gea capped a fine performance with another good save.
"It is a pity we did not take our chances," Ukraine coach Mykhailo Fomenko told a news conference.
"We had plenty of opportunities but their goalkeeper was brilliant tonight," he added.
(Additional reporting by Igor Nitsak in Kiev, writing by Iain Rogers, editing by Mike Collett and Martyn Herman)
