In this study, we aimed to study the effect of miR‐33b in regulating sensitivity to daunorubicin (DNR) in acute myelocytic leukemia (AML). We used quantitative real‐time polymerase chain reaction and Cell Counting Kit‐8 assay to detect the level of miR‐33b and cell viability. Cell apoptosis and the expression of eIF5A‐2 and MCL‐1 protein were detected by flow cytometry analysis and Western Blot analysis, respectively. MiR‐33b mimic increased sensitivity of AML cells against DNR, while miR‐33b inhibitor had the opposite effect. Furthermore, the results showed that the eIF5A‐2 gene was a direct target of miR‐33b, and miR‐33b regulated eIF5A‐2 mRNA and protein expression. Silencing of eIF5A‐2 by RNA interference increased the sensitivity of AML cells against DNR. We also found that MCL‐1 contributed to the regulation of DNR sensitivity, which was dependent on downregulation of eIF5A‐2. Finally, knockdown of eIF5A‐2 eliminated the effects of miRNA‐33b mimic or inhibitor on DNR sensitivity. These findings indicate that miR‐33b maybe as a new therapeutic target in AML cells.