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<span style="font-variant: small-caps;">d</span>-allulose has a significant application value as a sugar substitute, not only as a food ingredient and dietary supplement, but also with various physiological functions, such as improving insulin resistance, anti-obesity, and regulating glucolipid metabolism. Over the decades, the physiological functions of <span style="font-variant: small-caps;">d</span>-allulose and the corresponding mechanisms have been studied deeply, and this product has been applied to various foods to enhance food quality and prolong shelf life. In recent years, biotransformation technologies for the production of <span style="font-variant: small-caps;">d</span>-allulose using enzymatic approaches have gained more attention. However, there are few comprehensive reviews on this topic. This review focuses on the recent research advances of <span style="font-variant: small-caps;">d</span>-allulose, including (1) the physiological functions of <span style="font-variant: small-caps;">d</span>-allulose; (2) the major enzyme families used for the biotransformation of <span style="font-variant: small-caps;">d</span>-allulose and their microbial origins; (3) phylogenetic and structural characterization of <span style="font-variant: small-caps;">d</span>-allulose 3-epimerases, and the directed evolution methods for the enzymes; (4) heterologous expression of <span style="font-variant: small-caps;">d</span>-allulose ketose 3-epimerases and biotransformation techniques for <span style="font-variant: small-caps;">d</span>-allulose; and (5) production processes for biotransformation of <span style="font-variant: small-caps;">d</span>-allulose based on the characterized enzymes. Furthermore, the future trends on biosynthesis and applications of <span style="font-variant: small-caps;">d</span>-allulose in food and health industries are discussed and evaluated in this review.