The feasibility of T-Cell receptor (TCR) gene therapy using a MART-1-specific TCR has been previously demonstrated in melanoma patients. However, it remains a challenge without a defined tumor-specific antigen in the therapy of hepatocellular carcinoma (HCC). In this study, through the analysis of clonal expansion of TCR Vβ subfamily and DNA sequencing, we identified TCR Vβ7.1_H3F7 as a potential therapeutic gene specifically for the HCC patients. Peripheral blood monouclear cells (PBMC) transfected with TCRV β7.1_H3F7 gene were specifically cytotoxic against HCC cells in vitro. Adoptive transfer of this transfected PBMC resulted in a marked suppression of HCC tumor development in the animal model. These results demonstrated the value of TCRV β7.1_H3F7 as a therapeutic gene specifically for HCC. More importantly, it provides a novel strategy for screening tumor-specific TCR genes, which may pave the way for TCR gene therapy in cancer patients currently without the defined tumor-specific antigens.