Background: The aim of this study is to investigate changes of peripheral blood lymphocyte subsets before and after treatment with pembrolizumab for advanced oral cancer and its clinical sig-nificance. Methods: 32 patients with advanced oral cancer who received pembrolizumab treatment were selected as observation group, 30 healthy people during the same period were selected as control group. Before treatment and in cycles 1, 2, 3 and 4 after treatment, fluid cytometry was used to detect changes in levels of lymphocyte subsets in peripheral blood of patients. Results: CD3(+), CD4(+), CD4(+)/CD8( + )indexes of patients with advanced oral cancer before treatment were significantly lower than those in control group (P < .05), CD8( + )level was significantly increased (P < .05); After 1 cycle of pembrolizumab treatment, there was no significant difference in changes of lymphocyte subsets compared with before immunotherapy; After 2 and 3 cycles of treatment, CD3(+), CD4(+), CD4(+)/CD8( + )values were higher than before the treatment (P > .05), CD8( + )index was slightly lower than before treatment (P < .05); After fourth cycle of treatment, CD3(+), CD4(+), CD4(+)/CD8( + )values were significantly improved compared to before treatment (P < .05), CD8( + )index was significantly lower than before treatment (P < .05); In treatment process of patients with stable disease (SD)/partial response (PR), the CD3(+), CD4(+), CD4(+)/CD8( + )values of fourth cycles were higher than before treatment (P < .05), CD8( + )index was lower than before treatment (P < .05); During treatment of progressive disease (PD) patients, changes of lymphocyte subsets in fourth cycles were not significantly different from those before treatment (P > .05). This article shows through analysis that expression of programmed cell death ligand 1 (PD-L1) and pathological types have no obvious influence on effect of immunotherapy. Multi-factor analysis shows that it is more meaningful to observe the changes of CD3(+), CD4( + )and CD8( + )at the same time to predict effect of immunotherapy. Conclusion: Pembrolizumab can regulate changes of T lymphocyte subsets in patients with advanced oral cancer, improve immune status of patients, there is no obvious adverse reaction. Monitoring changes of lymphocyte subsets during treatment can predict effect of immunotherapy.