Objective The effect of different doses of ethylnitrosourea (ENU) and cyclophosphamide (CP) on the loss rate of CD59 on peripheral blood erythrocytes was explored to optimize the detection method of Pig-a gene mutation. Methods According to the weight and loss rate of CD59 on peripheral blood erythrocytes, rats were divided into 4 groups: the control group, CP 40 mg/ kg group, ENU 10 mg/ kg group and ENU 40 mg/ kg group (n =6). The control group was injected i. p. with PBS, other groups were injected i. p. with corresponding solutions. The body weight of rats on days 0, 7, 14, 21, 28, 42 and 56 were recorded. At the same time, blood samples were collected and incubated with antibodies, and the loss rate of RBC ^CD59- was detected by flow cytometry. Results Compared with the control group, at different time points, the body weight and weight gain of ENU 10 mg/ kg group and ENU 40 mg/ kg group had no statistically significant difference (P > 0.05), while those in the CP 40 mg/ kg group were significantly decreased (P <0.05). The loss rate of RBC ^CD59- was significantly increased in the CP 40 mg/ kg group at 28, 42 and 56 days, ENU 10 mg/ kg group at 42 and 56 days, and ENU 40 mg/ kg group at 7, 14, 21, 28, 42 and 56 days, (P < 0.05). The results showed a dose-response relationship. Conclusions Under the conditions of this Pig-a mutation detection method , ENU is superior to CP on raising loss rate of RBC ^CD59- , ENU 40 mg/ kg is better than 10 mg/ kg, and 28 days is suitable as the test period.