Panax ginseng is a traditional Chinese medicine with significant pharmaceutical effects and broad application. Rare ginsenosides with high antitumor activities can be generated via oriented modification of their glycosyl moiety. For this purpose, suitable microorganisms and their enzymatic systems can be used. In this review, we address several issues associated with these systems. Under aerobic conditions, fungus biotransformation provides an efficient and inexpensive biotransformation process that can be easily scaled up. Considering the profound use of probiotics, wild strains generally recognized as safe have shown a potential through classical fermentation in food manufacturers of deglycosylated ginsenosides. Commonly applied recombinant enzymes from E. coli, especially recombinant hyperthermophilic enzymes, showed efficient conversion in biomedical or pharmaceutical industries. In this review, key genes dedicated to the production of ginsenosides (especially in Saccharomyces cerevisiae) are highlighted in relation to the large-scale production of ginsenosides. We also evaluate biocatalytic strategies that are aimed to improve product specificity and biocatalytic efficiency with industrial applications. Perspectives of protein engineering and solvent engineering in the development and large-scale preparation of ginsenosides in anticancer drugs, food and health care products are explored.