Abstract：An immunosensor for direct amperometric determination of α-1-fetoprotein (AFP) was constructed by α-1-fetoprotein antibody (anti-AFP) immobilized on multilayers of horseradish peroxidase (HRP)/nano Au through electrostatic adsorption onto L-cysteine modified gold electrode surface. The fabricated procedures and electrochemical behaviors of proteins on such an interface were characterized with electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry. The determination of AFP was established by chronoamperometry to record the direct electrochemistry response of HRP to reduction of H2O2. Under optimal conditions, the immunosensor was highly sensitive to AFP with a detection limit of 0.5 ng•mL－1 and the linear range was at two concentrations from 1 to 10 and from 10 to 200 ng• mL－1 with 0.7 mmol•L－1 H2O2 in analyte solution. The dependence of the number of HRP/nano Au layer, pH, incubation time, reproducibility and stability was studied. More than 86.7% of the results of the human serum samples obtained by this method were in agreement with those obtained by radioimmunoassay.