Acute myeloid leukemia (AML) is a common hematologic malignancy in the elderly with frequent relapse and poor prognosis. Limited treatments highlight the need for novel natural anticancer compounds. Adlay, valued for its medicinal and dietary properties, exhibits anti-inflammatory and anticancer effects. However, research on adlay hulls, particularly their anti-AML bioactive molecules, remains insufficient. This study evaluated the effects of adlay hull ethanol extract (AHE) on AML cell proliferation and apoptosis. AHE was extracted with ethanol and fractionated using n-hexane, ethyl acetate, and n-butanol, followed by silica gel chromatography. Cytotoxicity was assessed via the CCK-8 assay, and mechanisms were analyzed by flow cytometry and Western blotting. The bioactive components were characterized by UPLC-IMS-QTOF-MS. AHE-EA-C (ethyl acetate fraction C) inhibited AML cell proliferation, induced G0/G1 phase arrest, and promoted apoptosis. It suppressed the PI3K/Akt pathway by reducing PI3K and Akt phosphorylation. Using UPLC-IMS-QTOF-MS analysis, a total of 52 compounds with potential anti-AML activity were identified in AHE-EA-C, among which neohesperidin and cycloartanol have been previously reported to exhibit anti-AML activity and thus hold promise as candidates for further development as AML inhibitors. This study is the first to identify adlay hull bioactive components and their anti-AML mechanisms via PI3K/Akt pathway inhibition, providing a foundation for developing natural anti-AML therapies.