This study was aimed to establish the technique of multiplex fluorescence in situ hybridization (M-FISH) and to explore its usefulness in detection of complex chromosomal aberrations (CCAs) in acute lymphoblastic leukemia (ALL). Five ALL patients with CCAs were analyzed by combining the techniques of conventional cytogenetics (CC) and M-FISH. The results demonstrated that M-FISH confirmed the aberrations previously detected by CC, such as t (9;22), t (1;19) and t (y;1), and revealed new abnormalities as der (1) (1::3::7), der (6) t (6;9) (q?;p13), der (1) t (1;11), der (12) t (1;12), der (3) t (3;5), der (2) t (2;16), der (9) (9::18::7) and der (7) (9::18::7), and also corrected the wrong results in CC. Among these abnormalities, der (9) (9::18::7) and der (7) (9::18::7) were reported for the first time. In conclusion, M-FISH has proved to be useful in characterization of the CCAs in ALL, and it is an essential method to refine the karyotype analysis.