To study the impact of T131N/M133T substitutions in hepatitis B virus surface antigen (HBsAg), the proteins expressed in Huh7 and HeLa cells were subjected to Western blotting, enzyme－linked immunosorbent assay (ELISA) and immunofluorescence (IF) staining. The results showed that T131N/M133T substitutions created a new site for N－glycosylation. The detected level of T131N/M133T was significantly low, indicating a potential negative impact on stability and antigenicity of HBsAg.