This study was aimed to investigate the immunophenotyping characteristics of biphenotypic acute leukemia (BAL) and to analyze its curative efficacy. The four-color direct immunofluorescence staining and CD45/SSC gating analysis of flow cytometry were used to detect the cells of bone marrow and peripheral blood of 45 doubtful cases of BAL, and the immunophenotyping was performed according to the European Group for the Immunological Classification of Leukemia (EGIL) score criterion. The results showed that among 45 doubtful cases of BAL, the co-expression of cCD79a and cMPO was observed in 23 cases of My/B-ALL, with highest level of CD19 expression, followed by CD10; the co-expression of cCD3 and cMPO was observed in 18 cases of My/T-ALL, with highest level of CD7 expression, followed by CD5; the co-expression of cCD3 and cCD79a was found in 4 cases of T/B. Out of 45 cases of BAL, 30 cases showed cTdT expression, 25 cases showed CD34 expression, 31 cases showed CD117 expression. The results also indicated that the expression rate of CD33 was higher than that of CD13 in antigen expression of My/B-ALL and My/T-ALL patients. The complete remission (CR) rate of My/B-ALL with My/T-ALL CD34(+) was lower than that of My/B-ALL with CD34(-), the CR rate of My/B-ALL with CD34(+) was lower than that of My/T-ALL with CD34(-). It is concluded that the co-expression of My/B-ALL antigen has been found to be most common, then followed by My/T-All. cCD3, cCD79a, cMPO have important value for diagnosing and identifying of BAL. The flow cytometry with four-color direct immunofluorescence staining is most specific method for diagnosis of BAL. The clinical prognosis of BAL with low cell differentiation is poor.