Carbohydrates play very important roles in a variety of physiological and pathological processes. Well-defined carbohydrates obtained by chemical synthesis are essential to the understanding of their functions. However, synthesis of carbohydrates is greatly hampered by its insufficient efficiency. We have developed the donor preactivation-based one-pot oligosaccharide synthesis strategy, and this strategy has been established as one of the efficient protocols for oligosaccharide assembly. Using this strategy, quite a lot of complex oligosaccharides with biological activities have been constructed successfully. On the other hand, assembly of longer carbohydrate chains remains one of the most challenging tasks for synthetic chemists. On the basis of oligosaccharide synthesis, we have realized a highly efficient assembly of a 92-mer polysaccharide, which is an essential structural constituent of mycobacterial cell wall, by the preactivation-based one-pot glycosylation protocol. Our work may open an avenue to the synthesis of complex polysaccharides with biological importance that are either difficult or impossible to access through isolation or semisynthesis.