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The 26-year-old's breathtaking Olympic debut with Valegro gained a standing ovation from a capacity 23,000 crowd at Greenwich Park.
Their Grand Prix score of 83.66 per cent was more than four percentage points better than the previous Olympic best of Germany's Kristina Sprehe that stood for just two hours.
The Team competition resumes and concludes next Tuesday with the Grand Prix Special test - Dujardin is world record-holder in that - with Britain having never previously won an Olympic Dressage medal.
Their current team score of 79.40 per cent - the average mark of all three riders Dujardin, Carl Hester and Laura Bechtolsheimer - is 0.56 per cent above Germany, with the Netherlands (76.80 per cent) third.
Dujardin's score was the best over two days of competition, with Dutch star Adelinde Cornelissen in second, Germany's Helen Langehanenberg third, Hester fifth and Bechtolsheimer seventh.
Barely 20 months ago, Dujardin had never ridden a competitive Grand Prix test, but today's performance has increased the prospect of double Olympic Games gold.
The Individual competition - freestyle to music - takes place next Thursday and closes the Olympic Equestrian schedule. The top 11 individual scores, plus all three members of the leading seven teams after today, go forward to the Grand Prix Special stage.