Hepatocellular carcinoma (HCC) is the most common type of liver cancer with limited treatments. Asia has the highest HCC incidence rates; China accounts for over 50% of all HCC cases worldwide. T-cell receptor (TCR) -engineered T-cell immunotherapies specific for human leukocyte antigen (HLA) -A*02:01-restricted α-fetoprotein (AFP) peptide have shown encouraging results in clinics. HLA-A*24:02 is more common than HLA-A*02:01 in Asian countries, including China. Here we identified a novel HLA-A*24:02-restricted peptide KWVESIFLIF (AFP ) located in AFP signal peptide domain by mass spectrometric analysis of HLA-bound peptides from HepG2 cells. A TCR (KWV3.1) specific for AFP -HLA-A*24:02 was isolated from peripheral blood mononuclear cells of a healthy donor. The binding affinity of soluble KWV3.1 to its antigen was determined to be ~55 μm, within the affinity range of native TCRs for self-antigens. KWV3.1-transfected T cells could specifically activate and kill AFP pulsed T2-A24 cells and AFP  HLA-A*24:02 tumor cell lines, demonstrating that AFP can be naturally presented on the surface of AFP tumor cell lines. The newly identified antigenic peptide can provide a novel target for immunotherapeutic strategies for patients with AFP  HLA-A*24:02 HCC.