Embodiments of the present disclosure relate to a D-amino acid oxidase mutant and application in preparing L-glufosinate thereof. The D-amino acid oxidase mutant has an amino acid substitution at at least one of position 62 and position 226 of an amino acid sequence of the D-amino acid oxidase mutant when compared to an amino acid sequence of a D-amino acid oxidase as set forth in SEQ ID NO. 1, the position 62 and position 226 being defined with reference to SEQ ID NO. 1, and the amino acid sequence of the D-amino acid oxidase mutant having at least 90% identity to SEQ ID NO. 1.