Abstract: Objective Laboratory animal model is an important tool in the field of biological drug development, including oncology research, antibody drug development and preclinical efficacy and safety evaluation. The aim of this study is to prepare CD40 humanized mice ( B-hCD40 ) and provide more possibilities for preclinical pharmacodynamics study and safety evaluation of CD40 antibody. Method B-hCD40 mice was constructed by CRISPER / Cas9 system. Then the expression of human CD40 was detected by RT-PCR and flow cytometry. A tumor model was well established basing on B-hCD40. Meanwhile, anti-tumor efficacy and safety of CD40 activated monoclonal antibodies were evaluated. Result Human CD40 was only expressed in B-hCD40, and the ratio of different lymphocytes had no significant differences compared to wild-type mice. CD40 monoclonal antibody could significantly inhibit tumor growth. The weight loss of mice of the treatment group may be caused by the liver and kidney toxicity of the antibody. Conclusion In our study, B-hCD40 were successfully prepared, which is an effective animal model for evaluating the efficacy and safety of CD40 antibody drugs.