A novel and sensitive immunosensor has been developed by electro-depositing gold nanoparticles on to a Prussian Blue-modified glassy carbon electrode for determination of hepatitis B surface antigen (HBsAg). After the developed immunosensor was incubated with different concentrations of HBsAg samples at 37 degrees C for 15 min, the current response decreased with an increasing HBsAg concentration in the sample solution. The decreased percentage of the current was proportional to HBsAg concentration ranging from 2 to 300 ng HbsAg ml(-1) with a detection limit of 0.42 ng HbsAg ml(-1) (S/N = 3). Analytical results of 50 specimens using the developed immunosensor showed satisfactory agreement with those using ELISA, indicating the method to be a promising alternative for detecting HBsAg in clinical diagnosis.