Recently, substantial levels of acidic d-amino acids, such as d-aspartate and d-glutamate, have been identified in many organisms, from bacteria to mammals, suggesting that acidic d-amino acids have multiple physiological significances. Although acidic d-amino acids found in animals primarily originate from foodstuffs and/or bacteria, the d-aspartate-synthesizing enzyme aspartate racemase is identified in various animals. In eukaryotic organisms, acidic d-amino acids are primarily degraded by the flavoenzyme d-aspartate oxidase (DDO). DDO is found in multiple eukaryotic organisms and may play important roles in acidic d-amino acid utilization, elimination, and intracellular level regulation. Moreover, owing to its perfect enantioselectivity and stereoselectivity, DDO may be a valuable tool in several biotechnological applications, including the identification and quantification of acidic d-amino acids. In this mini-review, previous DDO reports are summarized and the potential bioengineering and biotechnological applications of DDO are discussed.