Objective: Acute mixed-lineage leukemia (AMLL) is characterized as the acute leukemia involved with acute myeloid cells and lymphoid cells at the same time. The AMLL is easily misdiagnosed because of a dual character involved with lymphoid and myeloid cells. At present, researches of AMLL in adults are more common. Only some are reported for children. Therefore, our aim was to study clinical characteristics of the childhood AMLL. Methods: From January 2000 to July 2009, 14 cases of AMLL children were selected by morphological and immunophenotyping methods from 185 cases of childhood acute leukemia admitted to the Department of Pediatrics, Tongji Hospital, Tongji Medical College of Science and. Technology of Huazhong University. Medical records of all AMLL cases were reviewed for clinical characteristics. Findings: Fourteen cases of AMLL were screened from 185 cases of acute leukemia by morphology, immunology, cytogenetics and molecular (MICM). The rate of childhood AMLL accounted for 7.57% of pediatric acute leukemia (AL) diagnosed in the research period; white blood cell count in most of the patients was normal, the average value being 31.0x10(9)/L in the first visit. In the 14 cases of AMLL, 8 cases were B-Ly+/My+, 2 cases were T-Ly+/My+B, and 4 were T+B-Ly+/My+. Among them, nine cases received treatment. Consequently, 6 cases reached complete remission (CR); 1 case had not complete remission; 2 cases did not complete the treatment. Conclusion: The diagnosis of AMLL should depend on the comprehensive evaluation of MICM. As there are still many problems concerning AMLL, it is very necessary that the research units collaborate with each other to improve the prognosis of childhood AMLL. The limitations and applications of the results are that they are only based on the patients of one hospital.