To study the acute Leukemia immunophenotype and its diagnosis value, three color direct immunofluorescece staining methods of flow cytometry and immunophenotype of antibody integration system were used for detection of 180 cases of acute leukemia. The results showed that patients with ALL expressed lymphocyte antigen, and 49.4% patients with ALL accompanied myeloid antigen; all patients with AML expressed myeloid antigen, and 43.2% patients with AML accompanied lymphocyte antigen. In conclusion, leukemia immunophenotyping by three-color direct immunofluore staining methods could define some particular types of leukemia with an important value in diagnosis, treatment and predicting prognosis of acute leukemia.