Braspennincx edged out New Zealand's Ellesse Andrews by 0.061sec to win an enthralling final, with Canada's Lauriane Genest taking bronze.
Victory continues an impressive showing on the bike in Japan for the Dutch, who now own eight cycling medals at Tokyo 2020 and the most golds with four.
Annemiek van Vleuten and Anna van der Breggen claimed gold and bronze in the women's time trial, with Tom Dumoulin taking silver in the men's event and Van Vleuten also winning silver in the women's road race.
On the track, the Dutch quartet led by Harrie Lavreysen beat Britain on Tuesday to win the men's team sprint and there were gold and bronze medals for Niek Kimmann and Merel Smulders respectively in BMX.
Braspennincx won a silver medal in the keirin at the world championships in 2015 but she was not the pre-Games favourite for one of cycling's most unpredictable disciplines.
Invented in Japan in 1948, the keirin involves riders lurking behind a pacemaker before exploding in a sprint to the line in the last two-and-a-half laps.
Germany's Emma Hinze had been the favourite but she failed to go past the semi-final, finishing last in a race that Braspennincx won, laying the platform for her momentous gold medal.