To establish the reference interval (RI) of vitamin E for Chinese children and adolescents. Serum tocopherol samples were examined using high performance liquid chromatography in third-party clinical laboratory institutions. Using real-world data from multi-center clinical laboratory institutions in China, the distribution parameters of vitamin E levels were described and the RI was calculated using three algorithms. A total of 756,766 cases were included in the analysis, including 435,561 males and 321,205 females. The median of vitamin E in infants younger than 4 years of age initially increased but subsequently decreased; while its levels in children between 4 and 11 years of age remained relatively stable despite progressing in age (approximately 7.4–7.8 mg/L). After the start of puberty, the difference, relative to sex, gradually became apparent, and the median vitamin E levels in females was higher than in males. The differences of vitamin E levels between different regions and samples in different seasons had no clinical significance. The RI of vitamin E for children aged 0–18 years in China was 4.5–11.1 mg/L based on expectation–maximization algorithm. The RI established by the Hoffmann method was 4.6–12.8 mg/L. The age- and sex-specific RIs of vitamin E were established by an indirect approach. The RIs established by EM algorithms could be used as an alternative to establish RIs based on real-world data.