l-Ribose is a non-naturally occurring pentose that recently has become known for its potential application in the pharmaceutical industry, as it is an ideal starting material for use in synthesizing l-nucleosides analogues, an important class of antiviral drugs. In the past few decades, the synthesis of l-ribose has been mainly undertaken through the chemical route. However, chemical synthesis of l-ribose is difficult to achieve on an industrial scale. Therefore, the biotechnological production of l-ribose has gained considerable attention, as it exhibits many merits over the chemical approaches. The present review focuses on various biotechnological strategies for the production of l-ribose through microbial biotransformation and enzymatic catalysis, and in particular on an analysis and comparison of the synthetic methods and different enzymes. The physiological functions and applications of l-ribose are also elucidated. In addition, different sugar isomerases involved in the production of l-ribose from a number of sources are discussed in detail with regard to their biochemical properties. Furthermore, analysis of the separation issues of l-ribose from the reaction solution and different purification methods is presented. Key points center dot l-Arabinose,l-ribulose and ribitol can be used to producel-ribose by enzymes. center dot Five enzymes are systematically introduced for production ofl-ribose. center dot Microbial transformation and enzymatic methods are promising for yieldingl-ribose.