In this study, an interface embedded in situ gold nanoparticles (AuNPs) and biotin in chitosan hydrogel was constructed by one-step electrochemical deposition in solution containing tetrachloroauric (III) acid, biotin and chitosan. This deposited interface acts as biosensing platforms and provides specific binding sites for avidin, which are further capable of attaching any biotinylated bimolecular for biosensor design. Atomic force microscopy (AFM), A.C. impedance and surface plasmon resonance were used to characterize this interface. The immobilized acetylcholinesterase (AChE), as a model, showed excellent activity to its substrate and provided a quantitative measurement of organophosphate pesticide. Under the optimal experimental conditions, the inhibition of dimethoate was proportional to its concentrations in the range of 0.05 to 15 microg mL(-1) with detection limit of 0.001 microg mL(-1). The simple method showed good fabrication reproducibility and acceptable stability, which provided a new avenue for electrochemical biosensor design.