A novel photoelectrochemical biosensor incorporating nanosized ZnO semiconductor crystals with enzyme to enhance photochemical reaction has been investigated. In this work, the ZnO nanoparticles and acetylcholinesterase (AChE) were immobilized on Pt electrode by chitosan (CHI) via layer-by-layer technique (LbL) to fabricate a biological-inorganic hybrid system. Micrographs of (ZnO/CHI/AChE) films were obtained by scanning electron microscope, and photoelectrochemical properties of the resulting biosensors were measured by a three electrodes system and an ultraviolet lamp. Under ultraviolet light, the photo-effect of the ZnO nanoparticles showed enhancement of the biosensor to detect pesticide. Based on the inhibition of organophosphate pesticides on the AChE activity, using malathion 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.