Ascorbic acid (AA) is an esstential micronutrient in the human body and participates in vital processes. It can only be supplemented by external intake, making the determination of AA in food or beverages of significance. In the present work, MnO₂ nanoparticle (MnO₂ NP) based turn-off colorimetry was established for AA determination on the basis of AA-stimulated inhibition of catalytic oxidation. MnO₂ NPs were synthesized through a biotemplating method and displayed effective catalytic oxidation activity for oxidation of TMB, triggering a chromogenic reaction. Interestingly, the introduction of AA induced the decomposition of MnO₂ NPs and as a consequence decreased the colour. Therefore, sensitive and selective turn-off colorimetry for AA detection was established and a satisfactory linear range of 0.5–30 μM with a detection limit of 0.28 μM was obtained. This MnO₂ NP-based method was activated via a novel sensing principle, complementing and facilitating colorimetry for AA.