Accumulating studies have shown that miR‐216b acted as a tumor suppressor and was down‐regulated in solid tumors. However, little studies revealed the role or clinical implication of miR‐216b in blood cancers. Herein, we reported miR‐216b expression and its clinical significance in patients with acute myeloid leukemia (AML). In the current study, we analyzed bone marrow (BM) miR‐216b expression in 115 de novo AML patients examined by real‐time quantitative PCR. Notably, BM miR‐216b expression was significantly up‐regulated in AML patients, and could serve as a potential biomarker distinguishing AML from controls. No significant correlations of BM miR‐216 expression were found with sex, age, white blood cells, hemoglobin, platelets, BM blasts, French–American–British classifications, and karyotypes. Significantly, patients with high miR‐216b expression tended to have a lower frequency of FLT3‐ITD mutation and higher incidence of U2AF1 and IDH1/2 mutations. Moreover, complete remission (CR) rate and overall survival were negatively affected by BM miR‐216b overexpression among cytogenetically normal AML (CN‐AML). Cox regression analyses showed that high BM miR‐216b expression may act as an independent risk factor in CN‐AML patients. Among the follow‐up patients, BM miR‐216b level in CR phase was markedly lower than in diagnosis time, and was returned in relapse phase. Collectively, our findings indicated that miR‐216b overexpression was a frequent event in de novo AML, and independently conferred a poor prognosis in CN‐AML. Moreover, miR‐216b expression was a valuable biomarker correlated with disease recurrence in AML. Collectively, our findings indicated that miR‐216b overexpression was a frequent event in de novo AML, and independently conferred a poor prognosis in CN‐AML. Moreover, miR‐216b expression was a valuable biomarker correlated with disease recurrence in AML.