We developed a simple strategy for designing a sensitive electrochemical biosensor for organophosphate insecticides (OIs), based on an acetylcholinesterase (AChE)-carbon nanotube (CNT)-polypyrrole (PPy) biocomposite film modified gold electrode (labeled as AChE-CNT-PPy/Au). Based on the inhibition of OIs to the enzymatic activity of AChE, using malathion as a model compound, the conditions for detection of the insecticides were explored. The proposed method demonstrates an easy electron transfer between the immobilized enzyme and the electrode via functionalized CNTs in a polypyrrole matrix. Under optimal conditions, the inhibition of malathion was proportional to its concentration from 1.0-20.0 mu M. The detection limit was 0.1 mu M, with a good precision (RSD = 4.7 %, n = 8). The developed biosensor exhibited good stability. This method could be developed as a conventional one to select efficient enzyme inhibitors and investigate toxic compounds against enzymes.