Despite the tremendous advances made in the chemical glycosylation processes in the past few decades,the synthesis of biologically important complex carbohydrates is still challenging.An attractive alternative to chemical synthesis is enzymatic synthesis,which proceeds in high regio‐ and stereoselectivity without tedious protecting group manipulation.However,the enzymatic synthesis of complex carbohydrate is also challenging which involves an array of carbohydrate processing enzymes,especially different enzyme isoforms.Herein,we demonstrated that a number of complex biologically important carbohydrates can be generated in a highly efficient approach through rational design their in vitro biosynthetic pathway.