Abstract: Objective To investigate the levels of serum soluble vitamins A, D, and E in healthy children in Nanjing. Methods The data of physical examination of healthy children in the Second Affiliated Hospital of Nanjing Medical University were collected and grouped. The levels of serum vitamin A and vitamin E of each child were determined by reversed-phase high-performance liquid chromatography fluorescence detection. The levels of serum 25-hydroxy vitamin D (25(OH)D) were determined by liquid phase tandem mass spectrometry, and the levels of vitamins A, D, and E in each age group were analyzed. Results There were 2 151 children for health examination and the average serum vitamin A level was (0.32±0.08) mg/L, where the vitamin A level in the ≤1-year-old group was the lowest at (0.29±0.075) mg/L. The vitamin A detection rate was 37.09% in all children, and was the highest at 54.19% in the ≤1-year-old group. The average serum 25(OH)D level was (34.24±9.31) μg/L, and was the lowest at (25.58±8.67) μg/L in the >6-year-old group. The detection rate of moderate and low level 25(OH)D was 33.56% (722/2 151) in all subjects, and was the highest at 77.89% in the >6-year-old group. The average serum vitamin E level was (10.69±3.57) mg/L in all children, and was the lowest at (8.59±1.96) mg/L in the >6-year-old group. The detection rate of moderate and low level vitamin E was 5.57% (120/2 151) among all subjects, and was the highest at 20% in the > 6-year-old group. The levels of serum 25(OH)D and vitamin E in boys were lower than those in girls. The level of serum 25(OH)D in 6-year-< age ≤ 14-year-old boy group was lower than that of girl group (P=0.043). The level of serum vitamin E in the ≤ 1-year-old boy group and the 1-year-old Conclusions The vitamin A level in the ≤1-year-old group is the lowest. The levels of 25(OH)D and vitamin E in children gradually decrease as the increase of age.