A 3D ordered macroporous (3DOM) ionic-liquid-doped polyaniline (IL-PANI) inverse opaline film is fabricated with an electropolymerization method and gold nanoparticles; (AuNPs) are assembled on the film by electrostatic adsorption, which offers a promising basis for biomolecular immobilization due to its satisfactory chemical stability, good electronic conductivity, and excellent biocompatibility. The AuNP/IL-PANI inverse opaline film could be used to fabricate an electrochemical impedance spectroscopy (EIS) immunosensor for the determination of Hepatitis B surface antigen (HBsAg). The concentration of HBsAg is measured using the EIS technique by monitoring the corresponding specific binding between HBsAg and HBsAb (surface antibody). The increased electron transfer resistance (R-et) values are proportional to the logarithmic value of the concentration of HBsAg. This novel immunoassay displays a linear response range between 0.032 pg mL(-1) and 31 6 nor mL(-1) with a detection limit of 0.001 pg mL(-1) The detection of HBsAg levels in several sera showed satisfactory agreement wit a commercial turbidimetric method. h those using