The invention discloses a method for producing D-amino acid oxidase through fermentation, and belongs to the technical field of biological enzyme preparation. The method comprises the following steps: taking recombinant escherichia coli for expressing D-amino acid oxidase as a production strain, inoculating the recombinant escherichia coli into a fermentation culture medium for fermentation culture, supplementing materials in batches in a DO-STAT mode after dissolved oxygen is reduced to 1%, and setting the DO value to be 10-50%; in the fermentation process, adding 10-50 g/L lactose serving as an inducer, meanwhile, adding D-amino acid into the fermentation liquor, continuing culture till fermentation is finished, and collecting thalli containing D-amino acid oxidase in cells. In the fed-batch fermentation process, high-concentration lactose is added to serve as an inducer to improve the enzyme activity of DAAO, meanwhile, a proper amount of D-amino acid is added to reduce inhibition of DAAO on growth of host cells, the production efficiency of DAAO is greatly improved, and conditions are provided for industrial production.