The midfielder completed 82 minutes on his return to action after recovering from a knee injury sustained in this summer's European U21 Championships.
The German international was the catalyst for the turn around in the second half.
Always available, Holtby found space in between West Ham's midfield and defensive line and after creating a number of openings, smashed a spectacular 30-yard drive into the top corner to level the score on 66 minutes.
With just four minutes remaining, Ruben Lameiras provided a touch of ingenuity to unlock the home team's defence, allowing Shaq Coulthirst to volley home the winner.
Earlier, West Ham scored from corners on 23 and injury time of the first half to take a 2-0 lead.
Already on top in the second half, Simon Dawkins pulled a goal back on 56 minutes and there was no stopping the team from there as they made it three wins out of three so far in the Barclays U21 Premier League, with 13 goals already on the board. Indeed, after 31 games at U21 level, the team has only failed to score on two occasions.
The first half was a slow burner under the lights in East London.
Little happended in terms of goalmouth action until the 18th minute, when Paul McCallum latched onto Elliot Lee's pass and lobbed over the advancing Lawrence Vigouroux, but Zeki Fryers won the race to the loose ball against Blair Turgott.
Holtby's shot was blocked by Pelly Ruddock but it was soon 1-0 to the home side. George Moncur took a short corner to Lee, who drove across goal and Dan Potts was there to turn the ball home from close range.
Milos Veljkovic rifled just over from 25 yards and Cameron Lancaster broke into the box, only to be denied by George McCartney's block on the half-hour. McCallum then glanced a header inches wide.
It was warming up now. Coulthirst drew a fantastic save from Raphael Spiegel, the forward angling a reverse drive from the edge of the box that the goalkeeper kept out down to his right. West Ham broke immediately and Lee rolled a one-on-one just wide.
Holtby cracked in another effort straight at Spiegel in the 44th minute but a couple of late corners led to West Ham's second goal.
McCallum met Moncur's take from the right only for Coulthirst to slice off the line but from the resulting second corner, Ruddock headed the ball back into the danger area where McCallum planted home from eight yards.
Holtby took a leading role as we edged our way back into the game in the second half.
He set-up shooting chances for Coulthirst, who curled just over and Laste Dombaxe, who aimed just wide before blasting over himself from 25 yards.
The comeback was on after 56 minutes as Simon Dawkins drove through centrally before taking aim, his shot deflecting off McCartney to leave Spiegel helpless.
He was soon in action again though as Holtby let rip from 30 yards, this time the goalkeeper made a scrambling save to his left.
Holtby was now running the show and levelled with a screamer on 66 minutes. The goal actually stemmed from another rasper from the midfielder that Spiegel turned aside full length to his left. Play continued, the ball was played back to Holtby who this time let rip into the top corner from fully 30 yards.
Holtby was involved in everything and next rolled a pass into the path of Dombaxe, who connected well from 25 yards but the ball was always rising.
Gaps started opening up for West Ham as we pushed for a winner and Vigouroux was forced into two fine saves in a minute. The first a reaction stop to block Moncur's drive on 75 minutes and then full length to turn away Turgott's drive.
Holtby's work was over in the 82nd minute when he was replaced by Kenny McEvoy and it took just four more minutes for the lads to dig out the winner.
It was another special goal as well as Lamieras, faced with a wall of defenders 20 yards out, dinked a sand-wedge of a pass into the path of Coulthirst, who smashed home a volley from 10 yards.
West Ham U21 (4-4-2): Spiegel; Chambers, McCartney (Driver, 63), Ruddock, Potts; Turgott (Tombides, 88), Moncur, Whitehead, Lletget; Lee, McCallum (Sadlier, 59). Subs not used: Howes, Nasha.
Spurs U21 (4-2-3-1): Vigouroux; A McQueen, Ball, Fryers (Lameiras, 64), Stewart; Dombaxe, Veljkovic; Dawkins, Holtby (McEvoy, 82), Coulthirst; Lancaster (D McQueen, 64). Subs not used: Miles.