An amperometric biosensor based on screen-printed electrodes (SPEs) was developed for the determination of organophosphorus pesticides in water samples. The extent of acetylcholinesterase (AChE) deactivation was determined and quantified for pesticide concentrations in water samples. An enzyme immobilization adsorption procedure and polyacrylamide gel matrix polymerization were used for fabrication of the biosensor, with minimal losses in enzyme activity. The optimal conditions for enzyme catalytic reaction on the SPEs surfaces were acetylthiocholine chloride (ATChCl) concentration of 5 mmol/L, pH 7 and reaction time of 4 min. The detection limits for three organophosphorus pesticides (dichlorvos, monocrotophs and parathion) were in the range of 4 to 7 microg/L when an AChE amount of 0.1 U was used for immobilization.