Hepatitis B virus (HBV) is a small hepatophilic DNA virus that can specifically infect human hepatic parenchymal cells. The susceptibility of HBV can be attributed to cell surface receptor(s), intracellular proteins and other factors. A simple and clear method to assay the susceptibility of cells for HBV remains a challenge. Here, we constructed the recombinant HBV vectors 5c3c-CD59-Flag-GPI and 5c3cT-Flag-GPI based on the original HBV 5c3c vector and glycosylphosphatidylinositol (GPI) anchor. After transfection into Huh7 cells, Flag tags can be anchored on the cell membrane, and sorted by flow cytometry using Flag antibody. With the replenishment of HBV envelope protein, 5c3cT-Flag-GPI can form complete recombinant HBV particles. This study laid a foundation for optimization of packaging efficiency of recombinant HBV, which would provide a tool for HBV susceptibility research.