Provided is a D-amino acid oxidative enzyme mutant. The sequence of the mutant comprises a sequence by mutating the 54th amino acid residue N, the 58th amino acid residue F, the 211th amino acid residue C, and the 213th amino acid residue M of the sequence shown in SEQ ID NO:1 or the sequence having at least 76% identity with SEQ ID NO:1. The D-amino acid oxidative enzyme mutant has a higher enzyme activity, enzyme activity stability and/or ammonium resistance than a mild D-amino acid oxidative enzyme mutant. Also provided is an application of the D-amino acid oxidative enzyme mutant in preparing 2-oxo-4-(hydroxymethylphosphinyl)butyric acid.