To investigate the effects of CD3/CD28 antibody activation duration on CD8⁺ T cell phenotype and lentiviral transduction efficiency, naïve CD8⁺ T cells were activated with CD3/CD28 antibodies for 24 h, 48 h, or 72 h. Phenotypic changes including cell proliferation status and the expression levels of surface molecules CD44, CD62L, and CD69 were detected by flow cytometry. The activated cells were then infected with lentivirus, and transduction efficiency at different activation time points was evaluated using both live-cell imaging and flow cytometry. The results showed that the proliferation rate of CD8⁺ T cells in the 24-hour group was significantly higher than that in the 48-hour and 72-hour groups. For surface molecule ex-pression, the expression level of CD44 in the 24-hour group was significantly higher than that in the non-activated group. When the activation duration extended to 48 h, CD44 expression was further significantly upregulated, but showed no significant difference to that in the 72-hour group. CD62L expression remained at a relatively high level before activation, and was slightly downregulated after 48 h of activation, but re-mained stable among the 0-hour, 24-hour, and 72-hour groups. The expression of CD69 in the 24-hour group was significantly higher than that in the non-activated group, but there was no significant difference a-mong the 24-hour, 48-hour, and 72-hour groups. For lentiviral transduction efficiency, both the 48-hour and 72-hour groups showed a significant decrease compared with the 24-hour group. This study demon-strates that 24 h is an ideal activation duration, substantially improving CD8⁺ T cell proliferation, activation level, and lentiviral transduction efficiency upon CD3/CD28 antibody stimulation.