High expression of the Wilms' tumor gene (WT1) in acute myeloid leukemia (AML) has been considered as a sensitive marker of minimal residual disease (MRD). The present study investigated the significance of quantitative analysis of WT1 mRNA, combined with multiparameter flow cytometry (MFC) regarding its efficacy and prognostic as well as relapse prediction value for leukemia patients with hematopoietic stem cell transplantation. Reverse-transcription quantitative polymerase chain reaction analysis demonstrated that the expression of WT1 in the initial and relapse group was significant higher than that in the complete remission (CR) group (P<0.01). WT1 and the donor chimerism were negatively correlated (r=-0.73, P<0.05). In all AML patients, WT1 was the highest in the M3 subtype and the lowest in the M1 subtype. Follow-up of 12 AML patients demonstrated that WT1 gene expression levels markedly decreased after CR, but obviously increased after relapse, as did the rate of the leukemia cells detected by MFC. The combined usage of MFC and WT1 monitoring contributed to an improved detection rate of relapse (91.7%), and may be used to monitor MRD, assess the treatment efficacy and prognosis, and predict the risk of recurrence in leukemia patients without specific molecular markers after allogeneic hematopoietic stem cell transplantation.