ObjectiveTo establish the reference range of serum concentration of vitamin A (VA) and vitamin E (VE) in Southern Sichuan area of China.MethodsFrom August 1, 2021, to May 31, 2023, 9482 blood tablets were received for the screening of VA and VE. The information was divided into four different age groups: ≤1 year old, 1< to ≤6 years, 6< to ≤17 years, and 17< to ≤59 years. In each age group, the four seasons were further subdivided into spring, summer, autumn, and winter, as well as male and female genders. The serum concentration of VA and VE was detected by liquid chromatography—tandem mass spectrometry (HPLC‐MS), and the reference range was established for verification.ResultsThe concentration of VA and VE in 9482 cases showed skewed distribution. When comparing between different age groups, the serum concentration of VA and VE was statistically significant (p < 0.05). While comparing different seasons, the serum VA levels in different seasons were significantly different (p < 0.05) except in summer and autumn. There was statistical significance in VE level in different seasons (p < 0.05). And while comparing different genders, there was no statistical significance in VA concentration levels (p > 0.05). The VE concentration levels were statistically significant (p < 0.05). The established reference range was established and verified, and the results were in accordance with the standard.ConclusionThe reference range of VA and VE should be set according to different ages, different seasons, and different genders.