CD39, expressed by tumor infiltrating lymphocytes (TILs), is a marker to identify tumor-reactive T cells, which is frequently associated with stronger anti-tumor activity than bystander T cells in a variety of malignancies. Therefore, CD39 could be a promising marker for identifying the active anti-tumor immune cells used for cellular immunotherapy. To test this possibility, we constructed the hepatitis B virus surface protein (HBVs)-specific chimeric antigen receptor T-cells (HBVs-CAR-T cells) and generated the personalized tumor-reactive CD8 T cells. We subsequently assessed their anti-tumor efficiency mainly with a co-culture system for autologous HBVs-positive HCC organoid and T cells. We found that both CD39 HBVs-CAR-T and CD39 personalized tumor-reactive CD8 T cells induced much more apoptosis in HCC organoids. Although the exhaustion status of CAR-T cells increased in CD39 CAR-T cells, triple knockdown of PD-1, Tim-3, and Lag-3 with shRNAs furtherly enhanced anti-tumor activity in CD39 CAR-T cells. Furthermore, these CD39 CAR-T cells exerted an increased secretion of interferon-γ and stronger anti-tumor effect in patient-derived xenograft mouse model. Our findings demonstrated that CD39 could be a promisingly biomarker to enrich active immune cells and become an indicator marker for evaluating the prognosis of immunotherapy for HCC patients.