An amperometric acetylcholine biosensor was prepared by the generation of the conducting polymer poly(4-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)benzenamine) (poly(SNS-NH2)) on graphite electrodes. For pesticide detection, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were co-immobilized onto the conducting polymer poly(SNS-NH2) films using covalent binding technique. Electrochemical polymerization was carried out using a three-electrode cell configuration via cyclic voltammetry. Characterization of resulting acetylcholine biosensor was done in terms of optimum pH, enzyme loading, range of linear response and shelf-life. Linear range was 0.12-10 mM and shelf-life 4 weeks. Sensitivity was calculated as 2.19 mu A mM(-1) cm(-2). The designed biosensor was tested for the determination of paraoxon-ethyl in spiked tap water samples. The results were compared with a conventional quantification method using HPLC-DAD. Linear correlation of the quantification results with both methods (R-2=0.998) was obtained. (C) 2013 Elsevier B.V. All rights reserved.