Background: The reference intervals of biochemical markers were significantly affected by age and gender, espe-cially in minors. In recent years, many provinces and regions in China had established reference intervals for chil-dren's hematological indicators. Without considering the instruments and reagents, the reference interval will also be affected by the region, economic development, eating habits, and other factors. Therefore, the reference inter -val of any hematological indicators is not necessarily a fixed range, it changes with certain factors. In our study, we analyzed the changes of biochemical markers in different serum total Ca and 25-OH-D concentrations, and es-tablished the reference intervals of biochemical markers in 3-year-old children, explored the change trend of bio-chemical markers with different serum total Ca and 25-OH-D concentration. Methods: Data was collected from 226 cases of 3-year-old children for biochemical markers, in the Chinese PLA General Hospital in August 2015. The data were divided into a high-level group (serum total Ca > 2.63 mmol/L and 25-OH-D > 40.81 ng/mL) and a low-level group (serum total Ca < 2.54 mmol/L and 25-OH-D < 32.64 ng/mL) according to serum total Ca and 25-OH-D levels for comparison. The change trend of biochemical markers was compared according to serum total Ca and 25-OH-D level. Results: The Glu levels in boys were significantly higher than that in girls, but CHO and LDL-C in girls were sig-nificantly higher than that in boys. The reference intervals of ATL (5.6 -22.1 U/L), ALB (44.8 -55.2 g/L), TP (62.7 -83.1 g/L), ALP (154.4 -379.7 U/L), GGT (7.2 -15.9 U/L), Glu (boys: 4.08 -5.91 mmol/L; girls: 4.05 -5.37 mmol/L), UREA (2.7 -6.3 mmol/L), CREA (26.4 -46.8 mu mol/L), UA (182.4 -400.2 mu mol/L), TG (0.43 -1.67 mmol/L), CHO (boys: 3.19 -5.96 mmol/L; girls: 3.03 -6.51 mmol/L), HDL-C (0.98 -2.24 mmol/L), LDL-C (boys: 1.30 -3.64 mmol/L; girls: 1.24 -4.27 mmol/L), total Ca (2.34 -2.85 mmol/L), PHOS (1.38 -2.06 mmol/L), Mg (0.83 -1.06 mmol/L), osteocalcin (41.64 -91.92 ng/mL), PTH (12.08 -43.06 pg/mL), 25-OH-D (19.66 -56.37 ng/mL), beta-CrossLaps (0.82 -1.88 ng/mL), TP1NP (357.9 -1025.7 mu g/L) were established. ALT, TP, ALB, GGT, Glu, CHO, HDL-C, LDL-C, UREA, CREA, PHOS, Mg, and ALP in high level group were significantly higher than those in low level group. There was no significant difference in TG, UA, TP1NP, osteocalcin, PTH and beta-CrossLaps between high level group and low-level group. With the increase of serum total Ca and 25-OH-D levels, most of the biochemical markers had a gradually increasing trend. However, biochemical markers of bone (TP1NP, osteocalcin, PTH, beta-CrossLaps) showed different trends. Conclusions: This study established the reference intervals of biochemical markers of 3-year-old children. The changes of serum total Ca and 25-OH-D levels in children reflected the changes of glucose and lipid metabolism, liver and kidney function markers, and indirectly reflected the growth and development of children and various organ functions. Maintaining high levels of serum total Ca and 25-OH-D can promote the growth and develop-ment of children.