In the present study, a novel colorimetric sensing platform was constructed for quantitative detection of acetylcholinesterase (AChE) activity and its inhibitor. Manganese dioxide (MnO₂) nanosheets as an oxidase-mimicking nanomaterial could directly oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) into oxTMB without the need for horseradish peroxidase and H₂O₂. When AChE was introduced, acetylthiocholine could be catalytically hydrolyzed to produce thiocholine, which easily triggers the decomposition of MnO₂ nanosheets, causing the decrease of solution absorbance. Owing to the inhibition effect of organophosphorus pesticides, the enzymatic activity was suppressed, preventing the decomposition of MnO₂ and resulting in the increase of absorbance. Under optimal conditions, the colorimetric platform shows sensitive responses to AChE and paraoxon in the range of 0.1–15 mU mL⁻¹ and 0.001–0.1 μg mL⁻¹, respectively. The detection limits of AChE and paraoxon reached 35 μU mL⁻¹ and 1.0 ng mL⁻¹, respectively. Furthermore, the MnO₂-TMB platform has been used to fabricate test strips for rapid and convenient visual detection of AChE and its inhibitor with highly promising performance.