An electrochemical immunosensor has been developed for the determination of carbofuran based on gold nanoparticles (DpAu), prussian blue-multiwalled carbon nanotubes-chitosan (PB-MWCNTs-CTS) nanocomposite film and protein A (SPA) layer-by-layer assemble technology. DpAu and PB-MWCNTs-CTS incorporated films were used to enhance the electroactivity and stability of the immunosensor. The porous three-dimensional PB-MWCNTs-CTS nanocomposite film provided many amino groups and carboxyl groups to cross-link SPA and offered a large specific surface area to immobilize SPA. The formation of a self-assembled SPA layer was employed onto the electrode surface to increase the binding capacity of the antibody. The conformational process of the immunosensor and electrochemical performance of immunoreaction between carbofuran and the anti-carbofuran monoclonal antibody were characterized by cyclic voltammetry and electrochemical impedance spectroscopy, respectively. Effects of experimental variables, such as the concentration of the SPA and antibody, the adsorption time of DpAu, the pH of supporting electrolyte and the incubation time, were investigated in details. Under optimum operating conditions, the system provided a wide linear range between 0.1 and 1 μg/mL with a low detection limit of 0.021 ng/mL. The proposed immunosensor exhibited high sensitivity, high stability and good reproducibility, and it can be used for the detection of carbofuran pesticide.