Abstract: A novel amperometric biosensor for organophosphate pesticides based on multilayer films containing chitosan, multi-wall carbon nanotubes (MWNTs) and acetylcholinesterase (AChE) was developed. Micrographs of MWNTs/AChE multilayer films were obtained by scanning electron microscope, and properties of the resulting biosensors were measured by electrochemical measurements. Among the resulting biosensors, the biosensor based on five layers of multilayer films was best. Based on the inhibition of organophosphate pesticides on the AChE activity, using dimethoate as a model compound, the inhibition of dimethoate was proportional to its concentration ranging from 0.25 to 1.50 and from 1.75 to 10.00 μM, with a detection limit of 10 nM estimated at a signal-to-noise ratio of 3. The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.