Abstract: Alpha-fetoprotein (AFP) is expressed in approximately 70% to 80% of hepatocellular carcinoma (HCC) patients, serving as a target for immunotherapy. However, the clinical trial results of most AFP-based tumor vaccines have been unsatisfactory, so there is an urgent need to develop a more effective therapeutic tumor vaccine based on AFP antigen. This paper constructed an immunotherapy vector, namely Ad-hAFPm, using a replication-defective adenovirus as the vector. This vector aimed to achieve specific targeting of the AFP antigen by expressing a fusion protein of AFP and heat shock protein 70 (HSP70), along with granulocyte-macrophage colony-stimulating factor (GM-CSF). Both HSP70 and GM-CSF served as adjuvants to enhance the immunogenicity of tumor antigens. The therapeutic efficacy of the Ad-hAFPm vector was evaluated in xenograft models of two human AFP-overexpressing cell lines, with a non-targeted vector, Ad-hNYm, employed as a control. The results demonstrated significant tumor suppression in the Ad-hAFPm treatment group across both xenograft models. Specifically, there was a notable reduction in tumor growth rate, a decrease in tumor volume at the experimental endpoint, and a decline in serum hAFP protein secretion. These observations indicated the specific cytotoxic effects of Ad-hAFPm virus against AFP-positive tumor cells. Furthermore, enhanced infiltration of immune cells, particularly CD8⁺ T cells, was observed in the tumor tissues of the Ad-hAFPm group, suggesting that Ad-hAFPm virus could elicit an immune response against tumor cells.