Owing to their large specific surface, favorable electrical conductivity, and excellent electrocatalytic capabilities, two-dimensional transition metal dichalcogenides have received considerable attention in the field of biosensors. On the basis of these properties, we developed a portable and disposable enzyme-based biosensor for paraoxon detection using a metallic MoS2 nanosheets modified screen-printed electrode (SPE). The exfoliated ultrathin metallic MoS2 nanosheets can accelerate the electron transfer on electrode surface and contribute to the immobilization of acetylcholinesterase (AChE) via the cross-linking of glutaraldehyde. Electrodeposited gold nanoparticles (AuNPs) on SPE were used to immobilize MoS2 nanosheets through the interaction between Au atoms on AuNPs and S atoms on MoS2. Using acetylcholine as the substrate, AChE can catalyze the formation of electroactive thiocholine and further generate the redox current. In the presence of paraoxon, the activity of AChE can be inhibited, making the related electrochemical signals weaken. Under the optimized conditions, this electrochemical biosensor exhibited a favorable linear relationship with the concentration of paraoxon from 1.0 to 1000 μg L-1, with the detection limit of 0.013 μg L-1. Furthermore, this developed biosensor was successfully applied to detect paraoxon in pretreated apple and pakchoi samples, which can provide a reliable method for the rapid analysis of organophosphorus pesticides in agricultural products.