Background <p>Reliable interpretation of hematological and serum biochemical test results in donkeys benefits from the use of population- and management-system-adapted reference intervals (RIs). However, standardized and CLSI-compliant RIs for Chinese local donkeys are still unavailable, limiting their clinical and epidemiological application. This study aimed to establish hematological and serum biochemical RIs for Chinese local donkeys using a large, multi-farm dataset, and to evaluate the effects of farm origin, age, and sex on these parameters. Blood samples were collected from healthy donkeys raised under different conservation levels and production systems, and RIs were constructed following Clinical and Laboratory Standards Institute (CLSI) C28-A3 guidelines using non-parametric methods.</p> Results <p>A total of 1,163 serum samples collected from five geographically distinct donkey farms were included for serum biochemical analysis. The study population was sourced from national conservation farms comprising Dong’e and Wudi, provincial conservation farms comprising Yucheng and Zhongrun, and one local farm in Xinjiang. Twenty serum biochemical parameters covering protein metabolism, liver function, energy and lipid metabolism, mineral balance, and renal function were assessed. Hematological analyses were performed using samples obtained from 935 donkeys from the Dong’e farm. The results demonstrated that farm origin was the primary source of variation for most serum biochemical parameters. In contrast, although age- and sex-related differences were observed for certain variables, Harris and Boyd partitioning analysis indicated that these effects were not of sufficient magnitude to justify separate reference intervals for either biochemical or hematological parameters. Juvenile donkeys exhibited physiological trends consistent with growth and immune system maturation; however, these differences remained within the limits of non-partitioning criteria. Overall, the reference intervals established in this study differed from those reported in horses and donkeys from other regions, highlighting the substantial influence of population background and management conditions on physiological profiles.</p> Conclusions <p>This study provides the first large-scale, CLSI-compliant hematological and serum biochemical reference intervals for Chinese local donkeys. The findings indicate that population- and management-system-adapted reference intervals are important for accurate clinical interpretation. These reference values offer a useful baseline for veterinary diagnosis, health monitoring, and conservation management of Chinese donkey populations. </p>