Objective To prepare and evaluate a mouse model of chronic lymphocytic leukemia in order to provide a preclinical model for the treatment of chronic lymphocytic leukemia. Methods The cyclophosphamide (CTX)injection dose was determined after a literature review and experiments to prepare an immunosuppressed mouse model; 1 × 107 MEC-1 cells were transplanted into mice for three consecutive days by intraperitoneal injection and tail vein injection to prepare mice with chronic lymphocytic leukemia. Flow cytometry, immunohistochemistry, and other method were used to detect changes in peripheral blood leukocytes and CD19⁺ CD5⁺ B lymphocytes in mice with chronic lymphocytic leukemia and to analyze the infiltration of MEC-1 cells into the liver, spleen, thymus, lungs, lymph, and bone marrow. Results It was determined that a CTX dose of 150 mg/ kg injected for four days could significantly reduce the immune system function of mice. After transplantation with MEC-1 cells, mice exhibited chronic lymphocytic leukemia(CLL) characteristics. The number of white blood cells and B lymphocytes increased, and bone marrow cells multiplied. Conclusions A CTX dose of 150 mg/ kg injected for four consecutive days significantly reduced the immune function of mice and led to the successful preparation of a mouse model of CLL.