Objective: To explore changes in the percentage and cell absolute count of lymphocyte subsets in peripheral blood of patients with novel coronavirus pneumonia (COVID-19). Methods: The clinical data of 244 patients with COVID-19 admitted to the Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine from April 25 to May 16, 2022 were retrospectively analyzed and classified into difterent groups according to the types of diseases (mild, ordinary and severe), vaccination status and co-existing underlying diseases. Flow cytometry was used to detect the absolute counts and percentages of different lymphocyte subsets(CD3⁺T lymphocytes, CD3⁺CD4⁺T lymphocytes, CD3⁺CD8⁺T lymphocytes, CD16⁺CD56⁺NK cells and CD19⁺B lymphocytes) in each group. Further, we analyzed the relationship between the changes in peripheral blood lymphocyte subsets and the above clinical characteristics in patients. Results: Compared with those in the mild group, the absolute count of each lymphocyte subsets (P<0.05) and the percentage of CD3⁺T lymphocytes in the severe group were significantly decreased (58.0% vs 66.0%, P<0.05); Compared with the ordinary group, the absolute counts of CD3⁺T, CD3⁺CD4⁺T, CD3⁺CD8⁺T, CD19⁺B lymphocytes (P<0.05) were all decreased and the percentage of CD3⁺T lymphocytes in the severe group was decreased (58.0% vs 68.6%, P<0.05). Compared with those in the non-vaccinated group, the absolute counts of CD3⁺T (1 061/μL vs 858.2/μL), CD3⁺CD4⁺T (514.4/μL vs 645.1/μL), CD19⁺B (151.7/μL vs 249.6/μL) lymphocytes (P<0.05) and the percentage of CD19⁺B lymphocytes in the vaccinated group were increased (11.7% vs 15.4%, P<0.05). Compared with those without underlying diseases, the absolute counts of CD3⁺T (1 063/μL vs 891.4/μL), CD3⁺CD4⁺T (637.7/μL vs 540.3/μL), CD3⁺CD8⁺T (353.3/μL vs 299.5/μL), CD19⁺B (253.7/μL vs 154/μL) lymphocytes (P<0.05) and the percentage of CD19⁺B lymphocytes in patients with hypertension/diabetes were decreased (14.1% vs 11.8%, P<0.05). Conclusions: The changes in lymphocyte subsets in patients with COVID-19 are closely related to the severity of symptoms, vaccination history and history of underlying diseases. The decline in the level may indicate that the immune function of patients is reduced, and lymphocyte subsets measurement has essential reference value for the diagnosing, treating, and evaluating disease progression and prognosis in patients with COVID-19.