Objective: To establish an Epstein-Barr virus-transformed peripheral blood B cell line (BCL), and explore its phenotypic characteristics, the ability to secrete antibodies and cytokines, and the ability to present hepatitis B virus (HBV) antigen peptide. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with HBV infection. Epstein-Barr virus supernatant was incubated to construct BCL. The expression of CD19, CD138, CD38, CD27 and the production levels of IFN - γ, IL-10, IL-6 were detected by flow cytometry. BCL loaded with HBV antigen peptide was incubated with in vitro-expanded autologous T cells. Intracellular staining was used to detect the level of interferon-gamma produced by T cells. Results: Compared with untransformed peripheral blood B cells, BCL had high expression levels of CD138, CD38 and CD27, and the difference was statistically significant (P < 0.05), while the level of IL-6 production was decreased, and the difference was statistically significant (P < 0.01). BCL loaded with HBV antigen peptide had significantly enhanced the production of interferon-gamma by in vitro-expanded autologous T cells, and the difference was statistically significant (P < 0.01). Conclusion: BCL highly expresses CD138, CD38 and CD27, but its ability to produce IL-6 decreases. BCL can improve the immune response efficiency of HBV-specific T cells to HBV antigen peptide, and serve as a new tool for hepatitis B immune research.