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 especial 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.