<p>Rare sugars are monosaccharides and their derivatives that occur only in trace amounts in nature. Owing to their low caloric value and diverse physiological activities, they have attracted increasing attention as functional ingredients in the food, pharmaceutical, and health-related industries. However, their limited natural abundance and the inefficiency of traditional production methods remain significant challenges for large-scale application. Biosynthesis has emerged as a promising platform for rare sugar production due to its mild reaction conditions, environmental compatibility, and high catalytic specificity. With advances in synthetic biology and metabolic engineering, multiple biosynthetic routes have been developed. Further improvements in productivity and industrial feasibility increasingly depend on the coordinated integration of multi-level engineering strategies. In this review, we first briefly introduce the physiological functions and applications of representative rare sugars, including D-allulose, D-tagatose, D-allose, L-ribose, L-ribulose, and L-xylulose. We then provide a systematic overview of recent advances in biosynthesis, with a focus on metabolic engineering, enzyme engineering, biocatalysis, and fermentation process optimization. Finally, we discuss the key challenges and future directions for developing efficient microbial cell factories for rare sugar production.</p> Graphical Abstract <p></p>