A novel multilayer film based on Au nanoparticles (AuNPs) and polyaniline/carboxylated mul-tiwall carbon nanotubes-chitosan nanocomposite (PANI/MWCNTs/CS) was exploited to fabricate a highly sensitive immunosensor for detecting chlorpyrifos. PANI-coated MWCNTs were prepared by in situ chemical polymerization and carboxylated MWCNTs played an important role in obtaining the thin and uniform coat-ing of PANI resulting in the improved immunosensor response. AuNPs were used as a linker to immobilize chlorpyrifos antibody. The performance of the immunosensor was characterized by means of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM), respectively. All variables involved in the preparation process and analytical performance of the immunosensor were optimized. Under optimal conditions (antibody concentration: 5 μg/mL, working buffer pH:6.5, incubation time: 40 min, incubation temperature: 25℃), the immunosensor exhibited a wide linear range from 0.1 to 40 × 10?6 mg/mL and from 40 × 10?6 mg/mL to 500 × 10?6 mg/mL, and with a detection limit of 0.06 × 10?6 mg/mL, which provided a valuable tool for the chlorpyrifos detection in real samples.