It remains controversial whether tumour mutational burden (TMB) or neoantigens are prognostic markers in hepatocellular carcinoma (HCC). This study aimed to define the function of TMB or neoantigens in antitumour immunotherapy.Neoantigens of patients (n=56) were analysed by pVAC tools with major histocompatibility complex-1 (MHC-I) algorithms based on whole exome sequencing and neoantigens with mutant type IC50 <50 nM were defined as high-affinity neoantigens (HANs). Patients were segregated into HAN-high/low groups by median of HAN value, and overall survival (OS) was analysed. Autologous organoid killing model was developed to clarify the antitumour activity of HANs.The value of HAN showed a better correlation with OS (p=0.0199) than TMB (p=0.7505) or neoantigens (p=0.2297) in patients with HCC and positively correlated with the frequency of CD39+CD8+ tumour infiltrating lymphocytes (TILs). Furthermore, HAN-specific CD8+ T cells were identified in CD39+CD8+ TILs, which showed better antitumour activity in HAN-high versus HAN-low group. In addition, more effective HAN peptides were identified in HAN-high versus HAN-low group. Besides, flow cytometry data showed that in fresh tumour, CD39+PD-1intCD8+ TILs displayed an effector phenotype and stronger antitumour activity in HAN-high versus HAN-low group. More importantly, patients in HAN-high versus HAN-low group showed a better prognosis after anti-PD-1 therapy.Our study first demonstrates that HAN value positively correlates with better OS in patients with HCC. HANs trigger antitumour activity by activating tumour-reactive CD39+CD8+ T cells, and patients in HAN-high group benefited more from anti-PD-1 therapy than HAN-low group. These findings may provide a novel strategy for personalised antitumour therapies for HCC.