The objective of this study was to investigate the expression pattern of Wilms tumor 1 (WT1) gene at diagnosis, complete remission (CR) and relapse status in non-acute promyelocytic leukemia (non-APL) acute myeloid leukemia (AML) patients, and further explore the prognostic value of measurable residual disease (MRD) assessment by WT1 gene and multiparameter flow cytometry (MFC). Our results showed that the average expression level of WT1 was 4026 ± 616.1 copies/10 ABL at diagnosis, 155.3 ± 36.03 copies/10 ABL at CR, and 1766 ± 238.8 copies/10 ABL at relapse, with statistically significant differences ( = .000). ROC analysis showed that WT1 expression levels were 118.1 copies/10 ABL and MFC-MRD was 0.155%, which had good predictive efficacy for relapse of patients during consolidation therapy. Both WT1-MRD and MFC-MRD had a significant impact on relapse-free survival (RFS) and overall survival (OS). Patients with WT1-MRD positive or MFC-MRD positive were associated with worse RFS (HR 3.840, 95% CI 1.582-9.320,  = .003), (HR 4.464, 95% CI 1.841-10.984,  = .001) and worse OS (HR 2.963, 95% CI 1.058-8.295,  = .039), (HR 3.590, 95% CI 1.254-10.280,  = .017). Besides, compared with patients who were negative for both WT1-MRD and MFC-MRD, patients who were positive both WT1-MRD and MFC-MRD were associated with worse RFS (HR 6.200, 95% CI 2.206-17.430,  = .001) and worse OS (HR 4.886, 95% CI 1.388-17.197,  = .013). This study demonstrates that combined assessment of MRD by WT1 and MFC improves relapse and prognosis prediction in non-APL AML patients, and may help guide interventions for disease relapse.