United's youth team will face Prescot Cables at Brunton Park on Monday 13 November, kick off 7pm.
Prescot have been through three preliminary rounds to make it to the First Round Proper, beating Irlam Town, FC United of Manchester and Nelson FC.
United made it to the fourth round last season, beating FC United of Manchester, Chesterfield and Barnsley before being knocked out at the King Power Stadium against Leicester City.
Academy boss Darren Edmondson said: "We had a really enjoyable run in this competition last season and some of the lads will be remembering and looking back to that.
"The thing with this competition is that you tend to find out your opponents for the next stage in advance, and as we speak we know that round two will be against Hartlepool and round three is a chance to play Liverpool.
"Prescot know that as well, so they'll be going all out to get the victory as much as we will.
"It's a game on the main pitch, under the lights, and in front of a noisy crowd. The lads will have to manage their nerves but, if they get the ball down and play their game, they should be ok.
"We're looking forward to it and hopefully it will be a positive result."
Prices
Adults - £3
Concessions - £1
Tickets will be on sale from the west stand ticket office on the night.
