Acyl-coA oxidase (ACO) is an important flavoenzyme responsible for the first step of peroxisomal fatty acid β-oxidation. In this study, the roles of Tyr232 and Tyr401 in flavin adenine dinucleotide (FAD) binding and enzyme catalysis of ACO were explored using site-directed mutagenesis. For mutant proteins, different levels of activity loss were observed. Wavelength scanning of Y232 and Y401 mutant proteins indicated that there is no FAD binding in Y401S and Y401G mutant ACO. Structure analysis indicated that the phenolic hydroxyl and benzene ring of the side chain could stabilize FAD binding through hydrogen bonds network and hydrophobic pocket formation. These results indicated that these two tyrosine residues play a critical role in the FAD binding of ACO.