Reference intervals of biochemical tests for screening for diabetes mellitus and liver and renal function among school children in Central Taiwan have never been documented. Therefore, this study aimed to establish the reference intervals for the above mentioned biochemical tests for pediatric populations. Methods: A total of 4326 subjects, including 2029 kindergarten children, 1624 elementary-school children, 325 junior-high-school children, and 348 teachers were selected randomly in Central Taiwan. All serum alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (Cr) and glucose levels were determined using a Beckman Synchron CX5 analyzer. The reference intervals reflected estimates of the 2.5th–97.5th percentiles of non-parametric distributions. Results: Adults had significantly higher biochemical analyte values [except for BUN/creatinine (B/C) ratio] than children had. Multiple logistic regression analysis showed that biochemical analyte values were significantly higher in male than in female subjects. The concentrations of glucose and Cr increased with age. On the contrary, the B/C ratio decreased with age. Conclusion: Our study provides new pediatric reference intervals (2.5th–97.5th percentiles) of 60–99 mg/dL for serum glucose concentrations, 8–38 IU/L for ALT, 0.4–1.1 mg/L for Cr, 8.7–18.0 mg/L for BUN, and 10–34 for B/C ratio. The B/C ratio in children was higher than those of adults, possibily due to that children had a higher intake of protein.