Defoe slid in with five minutes left in extra-time as the visitors sensed another famous upset. It was harsh on Efetobore Sodje, who was magnificent at centre-back, and goalkeeper Darryl Flahavan, who denied Spurs with a string of saves.
The bottom side in the Championship had Kevin Maher and Mark Gower, former Tottenham players, in their starting XI and Gower offered a threat on the left in the early exchanges.
He whipped in an early cross, had a near-post effort saved by Paul Robinson and late in the first half curved a long-range effort wide of the upright as the underdogs started with energy, as they did in the last round when they defeated Manchester United.
Benoit Assou-Ekotto had Spurs first effort on goal, curling his free-kick around the wall but seeing the effort saved after Steed Malbranque had been tripped by Alan McCormack.
Spurs were inches away from breaking the deadlock midway through the first half from a move sparked by Danny Murphy playing in Defoe on the left.
The England striker found Mido and his volley smacked against the underside of the bar and away to safety.
Murphy also glanced a header wide while Flahavan, another hero from the United win, was at full stretch when Defoe leapt to meet Assou-Ekotto’s cross from the left.
Southend had a sight of goal on the stroke of half-time when Jamal Campbell-Ryce and Freddy Eastwood had efforts blocked on the edge of the area.
Predictably they had to withstand pressure after the break. Flahavan was forced into another save, four minutes into the second period, when Malbranque curled an effort from the edge of the area.
Defoe then came even closer when Sodje, an imposing figure throughout, misjudged a ball over the top but the England striker lobbed wide.
At the other end, Sodje was close with a header from a corner, with Eastwood almost getting on the end of the effort.
Jol then answered the home fans’ calls for Dimitar Berbatov to be introduced, and Murphy was taken off.
Berbatov, Dawson and Maher came close with extra-time efforts but it was Defoe who grabbed the winner.
In the 115th minute,Malbranque played the ballout to the left and Mido crossed invitingly for Defoe to slidein.
Eastwood could not direct a header when he was under the Spurs bar, and the hosts held on to book their place in Saturday’s draw.
TOTTENHAM: Robinson, Assou-Ekotto, Dawson, Tainio (Davids 56), Stalteri, Davenport, Malbranque, Huddlestone, Murphy (Berbatov 76), Defoe, Mido.
SOUTHEND: Flahavan, Hunt, Prior (Clarke 24), Sodje, McCormack (Francis 118), Campbell-Ryce (Hooper 114), Maher, Hammell, Gower, Eastwood, Bradbury.
Referee: P Dowd (Staffordshire).