Virus tagged with green fluorescent protein (GFP) contributes to the visualization and study of the virus in living cells. However, the hepatitis B virus (HBV) particle, which is a compact virion with limited internal space, cannot be incorporated with GFP tag as a large fragment. It was recently reported that protein genetically inserted with a smaller size tetracysteine (TC) tag could be specially labeled by a biarsenical fluorescent dye in living cells. In this study, we constructed a recombinant HBV vector encoding TC-tagged core protein for biarsenical labeling of HBV virion. TC tag was genetically inserted near the immunodominant c/e1 site of HBV core protein by mutagenesis. Western blot and enzyme-linked immunosorbent assay (ELISA) analysis showed that the TC-tagged core protein, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) could be expressed in cells transfected with the recombinant HBV vector, which is similar to the cells transfected with wild-type HBV vector. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis showed that HBV virion formation was affected by the genetic insertion of TC tag into core protein in some degree, but cells transfected with the HBV vector could still produce HBV virions incorporated with TC-tagged core proteins. Taken together, the recombinant HBV vector can serve as a useful tool to produce HBV virions incorporated with TC-tagged core proteins to be fluorescently labeled by biarsencial dye for visualizing and studying HBV in living cells.