The authors have synthesized molybdenum disulfide nanosheets (MoS nanosheets) by using a bottom-up hydrothermal method. The nanosheets display strong catalytic (enzyme mimetic) activity in catalyzing the oxidation of peroxidase substrate of 3,3',5,5'-tetramethylbenzidine (TMB) in presence of HO to produce a blue product. The peroxidase mimicking properties of MoS nanosheets depend on temperature, HO concentration and pH value. A choline assay was worked out where choline was oxidized by choline oxidase in presence of oxygen to produce HO which is colorimetrically detected, best at 652 nm. The method works in the 1 to 180 μM choline concentration range with a 0.4 μM detection limit. Color changes may also be detected visually. The assay is simple, highly sensitive, selective and rapid. It was applied in the determination of choline in (spiked) milk and serum. Graphical abstract Basic principle of intrinsic peroxidase-like activity of MoS nanosheets, applied to design a rapid and selective colorimetric assay for choline detection based on the tetramethylbenzidine (TMB) color reaction.