Lymphocyte subsets significantly change during childhood; thus, age-matched reference values derived from healthy children are crucial. We established reference values for lymphocyte subsets, including T cells (CD3+), CD4 T cells (CD3 + CD4+), CD8 T cells (CD3 + CD8+), double negative T (DNT) cells (CD3 + CD4-CD8-), B cells (CD3-CD19+), NK cells (CD3-CD56+), and NKT-like cells (CD3 + CD56+) in the peripheral blood of 813 healthy children. We used the method of the international standard document (Clinical Laboratory Standard Institute C28-A3) to establish reference intervals with a single platform. First, we used the Skewness and Kurtosis test to analyze the normality of the data. The nonnormally distributed data was transformed into approximately normal distribution by the Box-Cox transformation. Second, we used the Tukey's method to eliminate outliers. Further, all the subjects were grouped into subgroups according to sex (male and female) and age (0-1 month, 2-12 months, 1-3 years, 4-6 years, and 7-18 years). We used the standard normal deviation test (Z-test) to evaluate whether age and sex were possible grouping factors. The analyses indicated age to be an important factor associated with changes in lymphocyte subsets. The absolute number of lymphocyte subsets and total number of lymphocytes, T cells, CD4 T cells, CD8 T cells, and B cells gradually increase from birth to 12 months and then gradually decrease with age. Furthermore, CD4 T cells and the ratio of CD4+/CD8+ gradually decrease with age. In contrast, CD8 T and DNT cells gradually increase with age. The percentage and number of NK and NKT-like cells gradually increase with age and remain stable between 1 and 18 years of age. In conclusion, the age-related reference intervals established in healthy children in this study can aid in monitoring and assessing the changes in immune levels in diseased conditions.