Targeting oncogenic activating mutations of Fms-Like tyrosine kinase 3 (FLT3) has constituted a promising therapy for acute myeloid leukemia (AML). However, rapid development of resistance has significantly compromised clinical efficacy and therapeutic durability of FLT3 inhibitors. Covalent inhibitors have shown impressive potential in overcoming drug resistance. Herein, we designed and synthesized a series of 8-phenylquinazolin-2-amine derivatives as FLT3 covalent inhibitors targeting cysteine 828. Among them, 4k demonstrated potent and selective inhibitory activities against FLT3-ITD positive AML cells and BaF3 cells harboring drugresistant FLT3-ITD secondary mutations, including BaF3-FLT3-ITD-D835V/I. Biochemical and mass spectrometry analyses confirmed that 4k covalently bound to the Cys828 in the ATP pocket of FLT3. 4k also inhibited the phosphorylation of FLT3 and its downstream signaling factors, as well as induced cell cycle arrest and apoptosis. Furthermore, 4k, with an oral bioavailability of 12.48%, effectively suppressed tumor growth in a MV4-11 xenograft model without obvious toxicity. Taken together, 4k represents a novel covalent inhibitor targeting Cys828 of FLT3 kinase for targeted therapy of AML.