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Challenges in assessing hepatitis B virus (HBV)-specific T cell immunity as an immunological biomarker still remain in chronic hepatitis B (CHB), such as the requirement of large quantities of cells. This study aims to conveniently assess HBV-specific T cells immunity in chronic HBV infected patients. We obtained T cell receptor β chains (TCRβs) from public databases and six acute hepatitis B patients to establish an HBV-specific TCRβs dataset. For some TCRs from one acute patient, their specificities and epitopes were verified. The potential HBV-specific TCRβs from CHB patients were analyzed using GLIPH2 and established dataset. By analyzing two antiviral therapy cohorts including 42 CHB patients, we showed that individuals with better therapy response may depend more on newly emerging potential HBV-specific TCRβs. In a cross-sectional study containing 207 chronic HBV infected patients, the results exhibited that the characteristics of potential HBV-specific clusters were divergent between CHB and hepatocellular carcinoma patients. Our strategy could profile potential HBV-specific TCRβ repertoire using a small blood sample, which will complement traditional methods for assessing the HBV-specific T cell immunity. • A strategy for profiling the potential HBV-specific TCRβ repertoire using a small blood sample. • Chronic hepatitis B patients with better response to therapy depend more on new TCR. • Different HBV-specific TCRβ clusters characteristics between patients with chronic hepatitis B and hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]