Background This study aimed to analyze the lymphocyte subsets, their activities and their dynamic changes during immunochemotherapy in patients newly diagnosed with diffuse large B cell lymphoma (DLBCL). Methods Patients with DLBCL (n = 33) were included in the present study. Their peripheral lymphocyte subsets, phenotypes and functions were detected using flow cytometry. The dynamic results of lymphocyte activities were available for 18 patients. Results Compared with healthy controls (HCs), the counts of CD3(+), CD4(+), and CD8(+) T cells as well as those NK cells decreased in patients newly diagnosed with DLBCL, mainly attributed to patients with high risk of prognosis assessed by International Prognostic Index (IPI) score. Lymphocyte counts didn't present significant difference between high risk (IPI scores 3-5) and low risk patients (IPI scores 0-2), but CD4(+) T cells and CD8(+) T cells expressed higher levels of CD28 and HLA-DR, respectively, in patients with IPI score ranging from 3 to 5. Patients at high risk harbored higher percentage of regulatory T cells (Tregs), and their CD4(+) and CD8(+) T cells produced lower levels of IFN-gamma, reflecting an impaired cellular immune response. The dynamic changes of lymphocyte numbers and functions during treatment were further investigated. Total counts of CD3(+), CD4(+), CD8(+) T and NK cells progressively decreased because of the cytotoxicity of chemotherapy and then gradually recovered after six cycles treatment (rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisone, R-CHOP). The functions of CD4(+) and CD8(+) T cells recovered by the end of two cycles R-CHOP treatment, although NK cell function was not significantly affected throughout treatment. These results suggest that the counts and functions of lymphocytes are significantly decreased in patients with DLBCL, particularly those of CD4(+) and CD8(+) T cells. Conclusions The absolute counts and functions of CD4(+), CD8(+) T cells, which were significantly lower in patients with DLBCL, gradually recovered after effective treatment. Therefore, combined detection of T cell counts and functions are critically important for administering effective personalized immunotherapy as well as for identifying new prognostic markers or DLBCL.