Abstract: Objective To evaluate the specific cytotoxic effect of cytotoxic T lymphocyte (CTL) activated by peripheral blood monocyte cell (PBMC) loaded with hepatitis B core antigen (HBVc) -melanoma antigen gene-A3 (MAGE-A3) fusion protein on human non-small cell lung cancer cell with high MAGE-A3 expression in vitro. Methods PBMCs were isolated from peripheral blood of healthy individuals and stimulated with interleukin-2 (IL-2). The PBMCs loaded with HBVc-MAGE-A3 fusion protein were co-cultured with normal PBMCs to induce the specific cytotoxic T lymphocytes. NCI-H358 cell line with high expression of MAGEA3 antigen was selected as the target cell and HEK-293T cell line with low expression of MAGE-A3 antigen was used as the control cell to carry out the tumor cell killing test. Results Peripheral blood mononuclear cells (PBMC) loaded with HBVc-MAGE-A3 fusion protein antigen could induce specific cytotoxic T lymphocytes (CTLs), and it had significant cytotoxic effect on non-small cell lung cancer cells. The specific killing rate of experimental group was significantly higher than that of the control group (P< 0.05). In NCI-H358 cell group, when the effector target ratio (E:T) was 10:1, the killing rate of HBVc-MAGE-A3 group was about 60%, while it was about 35% in both HBVc group and NTC group with significant difference. When E:T=20:1, the killing rate of HBVc-MAGE-A3 group was about 90%, significantly higher than 45% in HBVc group and NTC group. And there was no significant difference between the three groups when using HEK-293T with low expression of MAGE-A3 as target cells. Conclusion Peripheral blood mononuclear cells loaded with HBVc-MAGE-A3 fusion protein antigen can successfully induce specific cytotoxic T lymphocytes (CTLs) and have specific immune killing effect on non-small cell lung cancer cells in vitro.