The clash will be played at Prenton Park on the weekend of January 4
Tranmere Rovers defeated Southport 2-0 on Monday night to set up a third round tie with Premier League side Tottenham Hotspur.
Two wonderful volleys from Connor Jennings helped knock out the sixth-tier side in their second-round replay.
A 1-1 draw at Prenton Park meant both sides knew victory would give them a home tie against Spurs.
And it was the League Two side who took the lead through Jennings’ sensational dipping effort from outside the box.
Jake Caprice put a good cross into the box and Jennings produced another spinning volley with his right foot before half time to secure the win.
Part-time Southport – ranked 59 places lower in the football pyramid – rallied after half-time but could not score.
Micky Mellon’s Tranmere side – who are eighth in the fourth tier – will now host Premier League Spurs in the opening tie of this season’s FA Cup third-round weekend on Friday, 4 January.