Objectives The Pediatric Reference Intervals in China (PRINCE) was initiated to establish the reference intervals (RIs) of Chinese children, as well as to make it possible to compare the variability of biochemical markers among countries internationally. Methods Healthy participants, aged up to 20 years, from 11 provinces across China, were enrolled in PRINCE and according to a standard screening procedure, that included a questionnaire survey, physical examinations and laboratory tests. Fasting venous blood specimens were collected. All serum specimens were analyzed with Cobas C702 in the center laboratory, i.e. clinical laboratory of Beijing Children’s Hospital, with certified qualification (ISO15189). The nonparametric method recommended by Clinical Laboratory Standards Institute guidelines, was used to calculate the age- and sex-specified RIs. Results Among the 15,150 participants enrolled, 12,352 children (6,093 males and 6,259 females) were included to calculate RIs. The RIs for total protein, albumin, globulin, calcium, phosphate, potassium, sodium, chlorine, alkaline phosphatase, γ-glutamyl transpeptadase, alanine aminotransferase, aspartate aminotransferase, creatinine and urea were established by age- or sex-partitions. Most biochemical markers displayed larger variability and higher dispersion during the periods between 28 days and 1 year old, and included 4–6 age partitions commonly during 1 to <20 years old. In addition, differences of RIs between sexes usually occurs around the initiation of puberty at 12–13 years old. Conclusions The age- and sex-specified RIs of 14 biochemical markers in PRINCE study can provide a solid reference, which will be transferred into relevant RIs for other clinical laboratory’s platforms according to the CLSI guidelines.