A novel gold nanorods (GNRs) biosensor based on the localized surface plasmon resonance (LSPR) behavior was designed to detect the hepatitis B surface antigen (HBsAg) which indicates active viral replication of hepatitis B virus (HBV). The surface of GNRs was modified with monoclonal hepatitis B surface antibody (HBsAb) through physical adsorption. Raman spectrum, dynamic light scattering (DLS) and zeta potential measurement were conducted to access the nature of the GNRs after antibody modification. The binding of analyte to the molecular probe was monitored by the longitudinal wavelength shift of LSPR peak in the UV-Vis extinction spectrum resulting from the changes of local refractive index induced by the immunological reaction. The biosensor could be utilized in quantitative analysis in Tris buffers, which has dose-dependence response ranging from 0.01 IU/mL to 1 IU/mL. Further, the biosensor was suited for qualitative analysis of HBsAg in the actual media of blood serum and plasma. The ease of operation, high sensitivity, and its generality offer specific advantages over other GNRs-based immunoassay methods.