Objective Comparing four groups of immunosuppressive mouse models established by cyclophosphamide administration in different doses and periods, we used partial least squares(PLS) analysis to evaluate the immunosuppressive mouse models comprehensively and select the appropriate way to establish this model. Methods In this experimental study, 58 male KM mice were randomly divided into five groups: normal group (10 mice) were given normal saline daily by i.p. injection, model group 1 (12 mice) was given 40 mg/kg CTX daily by i.p. injection for 10 days, model group 2 (12 mice) was given 80 mg/kg CTX daily by i.p. injection for 3 days, model group 3 (12 mice) was given 40 mg/kg CTX daily by i.p. injection twice for a week, model group 4 (12 mice) was given 50 mg/kg CTX daily by i.p. injection for 7 days. After the injection of cyclophosphamide, the daily metabolic activities were detected everyday such as body weight, water intake, and food intake, organ index and immunological indexes such as blood RT of the model mice were measured as well. Using partial least squares (PLS) analysis to the models and analyzing the final weight, final anal temperature, organ index, and blood routine examination in order to evaluate the immunosuppressive mouse models comprehensively. Results Compared with the normal group, different dosages of CTX reduced the weight and anal temperature of model mice (P<0.05), the food intake and water intake (P<0.01), and the spleen index and thymus index (P<0.01). Besides, the amount and percentage of basophilic granulocytes decreased (P<0.05), and the percentage of MCHC, macrophage went up, as well as the absolute value of WBC and lymphocytes were decreased in the model groups 1, 2, and 4 (P<0.05). According to the PLS analysis, there were significant differences among models 1, 2, and 4 when compared with the normal group, especially the most different in the model group 1. Conclusions After the PLS analysis of different indexes, the optimal way to establish immunosuppressive mouse models is the procedure with 40 mg/kg CTX daily injected i.p. for ten days. Our findings provide experimental evidence for the establishment of immunosuppressive mouse models.