Background: Immune checkpoint inhibitors (ICIs) have improved the therapeutic prospects in advanced gastrointestinal tumors. The PD-L1 expression has become one of predictive biomarkers for immunotherapy and used in treatment, but there are still some drawbacks. The aim of this study was to investigate the changes in peripheral blood T-lymphocyte subsets, IL-6 levels before and after treatment with ICIs and clinical significance. Methods: 70 patients with advanced gastrointestinal tumors who were hospitalized in Shaanxi Provincial People's Hospital from January 2020 to December 2022 were selected. 30 healthy patients were selected as controls. PD-L1 was detected by immunohistochemistry. Patients were treated with ICIs (sintilimab or pembrolizumab). 1 day before treatment, 20 days after the end of the 4 cycles of treatment, peripheral venous blood was collected and CT or MRI was performed to evaluate the therapeutic effect. Complete response (CR), partial response (PR), and stable disease (SD) were classified as immune response group, and progressive disease (PD) was classified as immune non-response group. To evaluate the effects of ICIs on the levels of T-lymphocyte subsets (Percentage of CD3⁺T cells, percentage of CD4⁺T cells, percentage of CD8⁺T cells, CD4⁺/CD8⁺, CD3⁺T cells, CD4⁺T cells, CD8⁺T cells) and IL-6. Results: Compared to controls, CD3⁺T cells, CD4⁺T cells, CD8⁺T cells and CD4⁺/CD8⁺ were significantly lower (P<0.05). After 4 cycles of immunotherapy, the CD3⁺T cells, CD4⁺T cells, CD8⁺T cells, and CD4⁺/CD8⁺ levels were significantly higher (P<0.05), and IL-6 levels were lower (P<0.05). After 4 cycles of treatment, 48 cases were divided in the immune response group and 22 cases in the immune non-response group, with a treatment efficiency of 68.6%. In the immune response group compared to the immune non-response group, the levels of percentage of CD4⁺T cells, CD4⁺/CD8⁺, CD3⁺T cells and CD4⁺T cells were significantly higher (P<0.05), while the percentage of CD8⁺T cells level was lower (P<0.05). The percentage of CD4⁺T cells, CD4⁺/CD8⁺, CD3⁺T cells, CD4⁺T cells and CD8⁺T cells were significantly higher (P<0.05) in the immune response group after 4 cycles of immunotherapy, while the percentage of CD8⁺T cells level was lower (P<0.05) compared to before immunotherapy. The percentage of CD4⁺T cells and CD4⁺/CD8⁺ were significantly lower (P<0.05), the percentage of CD8⁺T cells and CD8⁺T cells were higher (P<0.05) in the immune non-response group compared to before immunotherapy. In the immune response group, the percentage of CD4⁺T cells was higher in the PD-L1 high expression groups (≥ 50%) compared to the PD-L1 low expression groups (< 50%) (P<0.05). Conclusions: PD-1 inhibitors can affect the distribution of immune cells such as T-lymphocyte subsets in patients with advanced gastrointestinal tumors and improve the immune status of patients.