Objective To establish a method for in vitro expansion of cytomegalovirus-specific cytotoxic T lymphocytes (CMV CTL). Methods Full-length cytomegalovirus pp65 (CMV pp65) polypeptide 1 μg/mL was used to repeat rounds of in vitro stimulation of peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood stem cell collections mobilized by granulocyte-colony stimulating factor (G-CSF), and interleukin 2 (IL-2), IL-15 and IL-21 were also added for amplification for 20 days. On the 7th day of culture, autologous PBMCs loaded with CMV pp65 antigen peptide were added after being processed by mitomycin. On day 10, γ ray-radiated PBMCs loaded with CMV pp65 antigen peptide and CD3 (OKT3) were supplemented. For the control group, no antigen was added into PBMCs for amplification. The phenotypes of T lymphocytes and the secretion levels of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) before and after amplification were analyzed by polychromatic flow cytometry. The titer of serum CMV IgM/IgG antibody of the donors was detected by ELISA. Results (165.26±6.14)×106 cells were harvested after culture, including CD3+ T cells 89.21%. CD8+ T cells accounted for (43.54±28.03)% of CD3+ T cells, and CD4+ T cells for (34.23±26.18)%. The CD3+ T cells obtained were predominated by effector memory T cells (TEM), and the proportions of TEM in CD8+ T cells and CD4+ T cells after amplification culture were significantly higher than those before culture. Additionally, the proportions of stem cell-like memory T cells (TSCM) and tissue resident memory T cells (TRM) after culture were obviously raised than those before culture. In the functional experiment, the proportion of IFN-γ+ secreting CMV-specific CD8+ T cells after amplification was much higher than that before amplification, and the proportion of TNF-α+ CMV-specific CD8+ T cells was on the rise; the proportions of CMV-specific CD4+ T cells that could secrete IFN-γ and TNF-α had no obvious changes. Furthermore, the level of CMV IgG was positively correlated with the donor's ages, and in such a culture system, the proportions of amplified IFN-γ+ and TNF-α+ secreting CMV-specific T cells were negatively correlated with the donor's ages. Conclusion We have successfully established an effective method to amplify CMV CTL in vitro.