l-Rhamnose isomerase (L-RI, EC 5.3.1.14), catalyzing the isomerization between l-rhamnose and l-rhamnulose, plays an important role in microbial l-rhamnose metabolism and thus occurs in a wide range of microorganisms. It attracts more and more attention because of its broad substrate specificity and its great potential in enzymatic production of various rare sugars. In this article, the enzymatic properties of various reported L-RIs were compared in detail, and their applications in the production of l-rhamnulose and various rare sugars including d-allose, d-gulose, l-lyxose, l-mannose, l-talose, and l-galactose were also reviewed.