Objective CRISPR/Cas9 is used to construct the uricase-deficient mouse with stable inheritance, which is evaluated whether it can simulate the disease characteristics of hyperuricemia patients. Method Double sgRNAs were designed on both sides of Exon 2-4 of the Uox gene. The sgRNA and Cas9mRNA required for gene knockout was microinjected into the fertilized eggs of mice in a certain ratio. After 2-4 hours of cultivation, the embryos were transferred to surrogate mother mice to get F0 generation. PCR identification and sequencing analysis were performed on F0 mice to sift positive mice with the knockout of Uox gene. Then, positive mice were mated with wild-type mice for F1 generation. Heterozygous female and male mice of F1 generation were then selected to obtain homozygous F2 mice. Finally, the levels of uric acid, creatinine, urea, ALT, and AST in the serum of homozygous and wild-type mice were detected and compared. The pathological changes in kidney and liver tissues were observed through HE staining and Masson staining. Result Compared with wild-type mice, the levels of the uric acid (male mice: 478.4 ± 114.6 μmol/L, P<0.001; female mice: 507.7 ± 129.6 μmol/L, P<0.001), creatinine (91.8 ± 55.6 μmol/L, P<0.001), urea (28.6 ± 13.9 mmol/L, P<0.05), ALT (53.3 ± 23.3 U/L, P<0.01), and AST (203.3 ± 70.3 U/L, P<0.001) in the serum of Uox-/- ^mice were significantly increased. The histopathological results showed moderate hepatocyte degeneration in the liver, moderate to severe tubular cystic dilation, degeneration, and fibrosis in the kidney, glomerular hypertrophy and hyperplasia, small vessel dilation and congestion, and infiltration of stromal monocytes and lymphocytes for the Uox-/- ^mice. Conclusion The homozygous uricase-deficient mouse strain is successfully constructed by CRISPR/Cas9 technology, which is consistent with the relevant indicators of hyperuricemia patients and can be used as an animal model for research in the field of hyperuricemia.