Objective To collect the results of peripheral blood lymphocyte subsets determination conducted in National Center for Safety Evaluation of Drugs from 2007 to 2019 in accordance with Good Laboratory Practice (GLP), and establish reference ranges for corresponding normal values in BALB/ c mice, Sprague Dawley (SD) rats, and cynomolgus monkeys. Methods Peripheral blood samples were collected from animals and put into heparin sodium anticoagulant tubes. Blood samples from 303 BALB/ c mice, 359 SD rats, and 460 cynomolgus monkeys were stained with different combinations of fluorescent antibodies, and lymphocytes were classified and counted by their expression of CD3⁺ , CD4⁺ , CD8⁺ , and CD20⁺(cynomolgus monkeys only) using flow cytometry. For each species,the data were classified by gender and age (rat and mouse) and then calculated for normal reference range and 95% confidence interval, respectively. Results The range of CD4⁺ / CD8⁺ lymphocytes ratio was 1. 07-8. 59 in BALB/ c mice and 0. 54-11. 67 in SD rats. Ranges of CD4⁺ / CD8⁺ lymphocytes ratio and the percentage of CD3^- CD20⁺ lymphocytes in cynomolgus monkeys aged 3-4 years were 0. 35-4. 22 and 1. 40%-51. 99%, respectively. Conclusions Classified counts of lymphocyte subsets provide an important reference index for evaluating the immunotoxicity of biological products. The collection and establishment of relevant background data will facilitate more reasonable evaluations of drug toxicity.