d-Allulose, an epimer of d-fructose, is a rare monosaccharide that exists in extremely small quantities in nature. It is an ideal substitute for sucrose, because it has 70% of the sweetness of sucrose and ultra-low the energy. In addition, it has received sustained attention because of its unique physiological functions and potential health benefits. However, it is scarce in nature, and difficult to chemically synthesize. Because of its scarcity, bioconversion of d-allulose is attractive to researchers. It has been demonstrated that ketose 3-epimerase plays an irreplaceable role in the bioconversion of d-fructose to d-allulose. Herein, an overview of recent advances regarding the physiological functions as well as the biological production of d-allulose is provided. Additionally, a comparison of the biochemical properties and a structural analysis of ketose 3-epimerases are also reviewed in detail in this paper. Up to now, ketose 3-epimerase has been experimentally identified and characterized from only twelve types of microorganisms. In addition, 4 types of crystal structures from ketose 3-epimerases have been already solved, and the catalytic mechanism has also been proposed. However, the researches on molecular modification of ketose 3-epimerase are very few. In the future, molecular modification to improve the enzyme activity and thermostability through site-directed mutagenesis or directed evolution must be the research focus. •d-Allulose is an ideal substitute of sucrose, with high sweetness and low calorie.•d-Allulose is a rare sugar, with distinct physiological health functions.•Bioconversion of d-allulose using ketose 3-epimerase is the research focus now.•The enzyme properties are characterized and comprehensively reviewed.•The crystal structures are solved, and the active sites are explored.