Profiting from the rapid development of molecular biology technology, functional genomics plays an increasingly important role in analyzing microbial polysaccharide synthesis pathways. Various gene mining methods and genomics analysis tools facilitate the study of genes responsible for microbial polysaccharides synthesis. Here, we summarize the recent studies in biosynthetic pathway of versatile microbial polysaccharide biosynthesis and the key enzymes involved, the techniques and software package useful for gene mining and function verification. This review will provide a reliable technical reference for crucial genes verification in microbial polysaccharide synthesis and feasible solutions in strain engineering with elevating production titer.