A rapid, sensitive and quantitative assay method for Hepatitis B surface antigen (HBsAg) is of paramount importance for the drug development and in the diagnosis of this disease. Here, we proposed a novel biosensor that sensitively and selectively screen Hepatitis B surface antigen. This strategy relies on the cross-linking aggregation of gold nanoparticles (AuNPs) that were decorated with Hepatitis B surface antibody (HBsAb) and Raman reporter 5-thio-nitrobenzoic acid (TNB) by Hepatitis B surface antigen. The immune reaction between HBsAb and HBsAg offers this strategy high specificity, and the use of AuNPs additionally allows a visual and homogeneous assay format, thus permitting improved simplicity and throughput of the assays. The selectivity and sensitivity in HBsAg assay were achieved with a wide linear response range from 0.5 ng/mL to 50 ng/mL and a detection limit of 0.2 ng/mL. The results indicated that this strategy can offer a simple, robust and convenient platform for HBsAg analysis and related biochemical studies with high sensitivity and selectivity.