A highly sensitive potentiometric immunosensor for the diagnoses of epidemic diseases has been developed by means of self-assembly to immobilize hepatitis B surface antibody (HBsAb) for the detection of hepatitis B surface antigen (HBsAg) as a model. At first, the Nafion containing -SO3- groups was immobilized on a platinum electrode surface to absorb the -NH3+ groups of antibody molecules via the opposite-charged adsorption technique, in the meantime, hepatitis B surface antibodies were adsorbed onto the surface of Au nanoparticles, then hepatitis B surface antibodies and Au nanopartilces were entrapped into polyvinyl butyral on the surface of Nafion film. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited sigmoid curve with log HBsAg concentrations, high sensitivity, wide linear range from 26 to 1280 ng·mL-1 with a detection limit of 3.1 ng·mL-1, rapid potentiometric response (4 months). Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis.