A simple and cost-effective biosensor design is highly desirable for the detection of organophosphorus pesticides (OPs) due to their increasing environmental concerns. Mostly, OPs sensors rely on the inhibition of acetylcholinesterase activity. Also, most of the sensors exhibit a narrow linear range of OPs detection. Thus, to find an alternative for acetylcholinesterase based expensive sensors with simple and improved platform, we have designed choline oxidase (ChOx) inhibition based colorimetric sensor for OPs detection. The scheme operates through a distinct colorimetric output produced, upon oxidation of a redox sensitive dye, 2,2′-azino-di-[3-ethylbenzthiazoline sulfonate], by H₂O₂ generated upon oxidation by ChOx. In the presence of pesticide, paraoxon methyl, the enzymatic activity of ChOx is inhibited which reduces the colorimetric output. The current system records a LOD of 58 μM with a linear range of 0–18 mM. Additionally, to find mechanistic insights, molecular docking and molecular dynamics simulations have been performed. The response has also been demonstrated in real water sample.