Britain's Dujardin, who had appeared just once on her trusty stallion since their win at the London Games last summer, made light of their time apart at the second leg of the circuit in the Dutch city.
Dujardin, just 27, put on a remarkable display, scoring 82.191 per cent to claim victory ahead local hero Edward Gal aboard Glock's Undercover. The Dutchman finished second with a score of 79.660 per cent.
Sweden's Tinne Vilhelmson-Silfven completed the podium with Don Auriello after scoring 75.766 per cent.
Britain also topped the team classification with Daniel Watson/Fideramber, Carl Hester/Fine Time 13, Gareth Hughes/Dv Stenkjers Nadonna and Dujardin/Valegro edging out the hosts.
The Dutch team - made up of Madeleine Witte Vrees/Blom's Wynton, Hans Peter Mindhoud/Glock's Tango, Patrick van der Meer/Uzzo and Gal/Glock's Undercover - had to do with second place in front of their home crowd.
Sweden - Mads Hendellowitz/Weihenstephaner, Cecilia Andrén Dorselius/Lennox and Tinne Vilhelmson-Silfven/Don Auriello - came in third.
Despite the British dominance in Rotterdam, the Netherlands still lead in overall Nations Cup standings ahead of the third leg in Aachen, Germany, from June 25 to 30.