Objective To detect the difference between the peroxidase (POX) by cytochemical staining and cytoplasm myeloperoxidase (cMPO) by flow cytometry in acute leukemia cells, and provide a more accurate basis for the classification of leukemia. Methods The positive rate of POX in acute leukemia cells was detected by cytochemical staining. The positive rate of cMPO in acute leukemia cells was detected by flow cytometry. Then the positive rate of POX and cMPO, and the positive cells score were analyzed. Results The positive rate and the positive cells scores between POX and cMPO in acute lymphoblastic leukemia were significantly different (P<0.05), the positive rate and the positive cells scores of POX were significantly higher than those of cMPO. The positive rate between POX and cMPO in acute non-lymphoblastic leukemia (ANLL) had significant differences (P<0.05), the positive rate of cMPO was higher than that of POX; but no difference was found between POX and cMPO positive cells scores in ANLL (P>0.05). In acute myelocytic leukemia (AML)-M1 subtype, significant difference was found in the positive rate between POX and cMPO (P=0.006); cMPO positive rate was significantly higher than that of POX, but the POX positive cells score was significantly higher than that of cMPO (P=0.001). There were no significances of positive rate and positive cells score in AML-M2, AML-M3, AML-M4, AML-M5 subtypes between POX and cMPO (P>0.05). Conclusions There are not major differences between positive rate of POX and cMPO, as well as the positive cells scores in acute leukemia, especially acute myelocytic leukemia. We can choose the better method according to the actual situation and the sensitivity requirements. The two methods should be replenished by each other and used alternately.