This paper describes the construction, evaluation, and application of an electrochemical biosensor for the determination of carbaryl. The biosensor was constructed using the self assembled monolayer technique with gold substrate modified with nanostructured monolayer of fourth-generation poly(amidoamine) with a cystamine core (PAMAM-G4) and covalent immobilization of acetylcholinesterase enzyme (AChE). The surface coverage was investigated by cyclic voltammetry and linear voltammetry. The results of partial surface coating and electrochemical desorption showed that the studied time interval of 12 h is necessary to form and stabilize the monolayer time. The biosensor electrochemical properties were investigated using cyclic voltammetry and electrochemical impedance spectroscopy. Parameters affecting the biosensor response such as enzyme amount, concentration of glutaraldehyde, and inhibition time were optimized. Based on the inhibition effect of carbaryl on the AChE activity, this biosensor showed a linear range from 1.0 at 9.0 mu mol L-1 and detection limit of 0.032 mu mol L-1.