L-ribose is a non-natural and expensive sugar that can be used as an important intermediate for the synthesis of L-nucleoside analogues, which are used as antiviral drugs. In contrast to chemical production, biotechnological methods can produce L-ribose from biomass under environmentally friendly conditions. In this mini-review, various strategies for synthesizing L-ribose by applying microorganisms and their enzymes are discussed, including microbial biotransformation and biocatalysis by engineering bacteria. Furthermore, subsequent isolation-and-purification techniques, as an integral step in the whole process, are accordingly described, containing the special introduction of a promising strategy of L-ribose separation. Particularly, further researches and outlook for the improvement of L-ribose preparation was solely stressed. Compared with each method, this mini-review provides a panorama of respective advantages and disadvantages existing in them.