〔Abstract〕 Objective To construct an Il-9 (Il9) gene knockout mouse model on a C57BL/6N background, establish a reliable genotyping method, and provide a standardized animal model for investigating Il9-related immune mechanisms and diseases. Methods Il9 knockout mouse strain (C57BL/6N-Il9em1Cya) was generated using conventional gene knockout technology. The method for DNA extraction from mouse tails was optimized to ensure template quality. Two sets of specific primers (F1/R1 and F1/R2) were designed to amplify target fragments by PCR, and mouse genotypes were determined by agarose gel electrophoresis. Specifically, primer pair F1/R1 targeted the knockout sequence with an expected product of 465 bp, while F1/R2 targeted the wild-type sequence with an expected product of 703 bp. Subsequently, Western blot was performed to verify the knockout efficiency of Il9 in primary immune cells and key organs of the offspring mice, and flow cytometry was used to assess the effects of Il9 deficiency on the function of the immune system in major immune cell populations. Results Il9 knockout mice were successfully bred and genotyped. PCR identification showed that homozygous mice (-/-) exhibited only a 465 bp band, wild-type mice (+/+) displayed only a 703 bp band, and heterozygous mice (+/-) showed both bands simultaneously. Meanwhile, Western blot analysis revealed significantly decreased Il9 expression in various organs of the offspring mice (P<0.001). Flow cytometric results demonstrated that Il9 deficiency exerted no obvious effects on the proportions of peripheral blood lymphocytes, splenic lymphocytes, and peritoneal macrophages. Conclusion An Il9 gene knockout mouse model on a C57BL/6N background is successfully constructed. The established PCR genotyping system is highly efficient and accurate, which can effectively distinguish mice of different genotypes. This model provides a stable and reliable experimental basis for subsequent functional studies of Il9 and exploration of the mechanisms underlying Il9-related diseases.