Abstract: Organophosphorus and carbamate pesticide residues pose severe threats to food safety, making it essential to develop efficient and rapid screening technologies. This study was based on the enzyme inhibition method, using the catalytic activity of acetylcholinesterase (AChE), with acetylthiocholine iodide (ATCI) as the substrate and 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) as the chromogenic agent, to prepare a one-step rapid test strip for pesticide residue detection. The test strip was constructed as follows: using a PVC plate as the supporting substrate, it integrated six key components sequentially, including the sample pad, enzyme pad, blocking pad, substrate pad, chromogenic pad, and adsorption pad. Chitosan nonwoven fabric was identified as the optimal immobilization material for the enzyme pad, substrate pad, and chromogenic pad. Optimization results indicated that the best immobilization conditions for the enzyme pad were pH = 8.0 and immobilization at 4 ℃ for 8 hours. Sensitivity tests demonstrated that the test strip exhibited significantly higher sensitivity to pesticides compared to free enzymes and commercially available kits, with half-maximal inhibitory concentrations (IC₅₀) for trichlorfon, phoxim, malathion, carbaryl and carbofuran reaching 0.2, 0.25, 0.35, 1.6 and 0.4 mg/L, respectively. The test strip enables detection within 15 minutes at room temperature, featuring both rapid detection and biodegradable properties. It remained stable for up to 60 days when stored at 4 ℃ away from light, making it well-suited for on-site testing at the primary level and demonstrating significant application value.