Objective To detect the effect of ethanol on T lymphocyte subsets, immune activity and apoptosis in peripheral blood. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by density centrifugation. Lymphocytes were co-cultured with phytohaemagglutinin (PHA) in 30 and 50 mL/L ethanol. At 12, 18, 36, 48 hours after PBMCs were exposed to alcohol, flow cytometry was performed to examine the alterations of T lymphocyte subsets. Results Compared with the control group, at 12 hours after the exposure, the number of CD8+CD69+ T lymphocytes increased significantly in both PHA and alcohol groups but the number of CD8+CD69+ T lymphocytes in the PHA group was apparently higher than that of 50 ml/L ethanol group, while the number of T cells had no obvious change. At 18 hours after exposed to ethanol, the proliferation of T lymphocytes was reduced obviously in the ethanol groups. In addition, the proliferation of T cells in the ethanol (50 ml/L) group was markedly lower than that in 30 ml/L ethanol group. At the same time, the number of CD8+CD69+ T cells was markedly lower. At 36 hours after exposed to ethanol, in comparison with the PHA group, the number of T lymphocytes decreased markedly in the ethanol groups. At 48 hours after exposed to ethanol, T cells almost disappeared in the ethanol groups. The apoptosis rate of CD3+ T lymphocytes increased with the extension of co-culture time of PBMCs and ethanol as well as the increase of ethanol dose. Conclusion With the increase of ethanol dose, the activity of T lymphocytes in the peripheral blood treated with higher dose of ethanol would go down and the number of T lymphocyte subsets and the function of T cells decrease, which might be related to the ethanol that promotes the apoptosis of T lymphocyte subsets.