A novel amperometric biosensor based on a conducting polymer using multi walled carbon nanotube modified electrode was developed for detection of organophosphorus pesticides. Acetylcholinesterase (AChE) was successfully immobilized by covalent linkage on the modified graphite electrode. Carbon nanotubes were functionalized by electrochemical treatment. A conducting polymer; poly(4-( 2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)benzenamine) (poly( SNS-NH2)) was synthesized via electropolymerization to examine its matrix properties for biomolecule immobilization. This strategy enhanced electron transfer rate at a lower potential (+100 mV vs. Ag reference) and catalyzed electrochemical oxidation of acetylthiocholine effectively. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), contact angle measurements and electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) techniques were used to monitor changes in surface morphologies and electrochemical characterizations. The proposed biosensor design offered a fast response time (6 s), a wide linear range (0.05 mM and 8.00 mM) and a low detection limit (0.09 mM) with a high sensitivity (24.16 mu AmM-1 cm(-2)) for acetylthiocholine. The inhibition responses of paraoxon, parathion and chlorfenvinphos on the enzymatic activity of AChE were detected. The fabricated biosensor was tested for the detection of pesticides in fortified tap water samples. The results were found to be in good agreement with the ones determined by HPLC/DAD technique. (C) 2014 Elsevier B.V. All rights reserved.