In this study, multi-walled carbon nanotubes (MWCNTs), gold nanoparticles (GNPs) and Prussian Blue (PB) were used for modifying glassy carbon working electrode (GCE) to construct acetylcholinesterase (AChE) biosensor respectively. Chitosan membrane was used for immobilizing AChE through glutaraldehyde cross-linking attachment to recognize pesticides selectively. Before the detection, the enzyme membrane was quickly fixed on the surfaces of modified electrode with O-ring to prepare an ampero-metric acetylcholinesterase biosensor for organophosphate pesticides. The fabrication procedures were characterized by cyclic voltammetry and amperometric i-t curve. The electrochemical behaviours of three modified sensors were compared, and the results showed that AChE-PB/GCE possessed higher oxidation peak current at a lower potential. Based on the inhibition of organophosphorus pesticides to the enzymatic activity of AChE, using dichlorvos as model compound, the sensitivity of three modified biosensors were compared, the results showed that the detection limit of AChE-PB/ GCE was lowest.