Due to their specific spinel structure, ternary oxides with multi-catalytic sites on a highly active exposed surface are recommended as alternative bio-catalysts. Spinel zinc vanadate with two-dimensional nanosheets (Zn3V3O8 NSs) was synthesised using a one-step hydrothermal route with CTAB and glycine as a bi-surfactant, where each NS has a thin thickness (25 nm) and wide cross section (2 μm). As a key parameter for peroxidase-like activity, the Michaelis-Menten constant (Km) for Zn3V3O8 NSs was calculated to be 0.271 mM with TMB and 1.317 mM with H2O2 at optimum conditions, indicating a higher affinity for the exposed (011) facet towards horseradish peroxidases. This affinity is related to the geometric matching between V4+ active sites and the terminal amino groups of TMB. The V4+ ions on the (011) facet act as dangling bonds and readily react with H2O2 in a Fenton-like reaction. The peroxidase-like activity for Zn3V3O8 NSs is verified by the formation of [V(IV)-OO˙] by the ˙O2- and V5+ near V4+ sites, but oxidase activity for Zn3V3O8 NSs. Based on the peroxidase-like activity, Zn3V3O8 NSs were used as a colorimetric glucose sensor with a wide linear range from 0.01 to 0.5 mM and a detection limit (LOD = 3σ/S) of 2.81 × 10-7 M. The colorimetric sensor also exhibited high accuracy and selectivity in synthetic perspiration samples.