Ajax won 2-1, although it was not enough to see them through to the Last 16 of the Uefa Europa League as they lost down 3-2 on aggregate.
Serero, however, should be pleased with his 30 minutes against the English Premiership giants.
He replaced the injured Christian Eriksen after an hour and got stuck into the attack.
It was Serero's 10th appearance for his new club. He also made a substitute appearance for Ajax at Real Madrid in the Uefa Champions League earlier this season.
Ajax played a very similar team to the one that lost last week. Ricardo van Rhijn came into the side for Ismael Aissatti, while Nicolas Lodeiro started as part of the front three with Dmitry Bulykin missing out through injury.
Sir Alex Ferguson made four changes from the side that won in Amsterdam a week ago, with Rafael da Silva and Chris Smalling coming in for Jonny Evans and Rio Ferdinand, while in midfield, Park Ji-Sung replaced Michael Carrick in the centre.
Up front, Dimitar Berbatov replaced the ill Wayne Rooney as the Bulgarian started alongside Hernandez, with Danny Welbeck providing cover from the bench.
After an energetic start from both sides, Ajax often had long periods of pressure and possession, but it was the hosts who took the lead, with Javier Hernandez producing a calm finish to open the scoring, after Dimitar Berbatov slipped through a pass.
Aras Ozbiliz equalised for the Dutch side with a well taken half-volley and Toby Alderweireld scored the late winner with the match coming to a close, although the hosts managed to hold on.