Objective The ob mice (C57BL/6-Lepem1/Nifdc) with the Lep gene knocked out mice (ob/ob) were generated by CRISPR/Cas9 system, establishing an animal model suitable for preclinical drug evaluation in clinical diseases such as diabetes. Methods According to the principle of CRISPR/Cas9 target design, sgRNA targeted mouse Lep gene was designed for transcription in vitro, and micoinjected with Cas9 mRNA into mouse zygotes. Mice tails DNA was extracted and detected by PCR and sequencing, followed by mating positive mice and wild-type mice. Blood biochemistry and liver pathology were assessed in homozygous ob mice. Results 8 positive mice were identified and a stable mouse strain was selected for further breeding. The serum triglycerides (TG), total cholesterol (CHO) and alanine aminotransferase (ALT) levels in homozygous ob mice were significantly higher than wild-type mice. Liver pathology result showed significant inflammatory infiltration and lipid vacuolar transformations. Conclusion The successful establishment of Lep gene knockout mice has enriched the national rodent experimental animal database and provided an animal model for preclinical drug evaluation.