The expanding interest for carbohydrates and glycoconjugates in cell communication has led to an increased demand of these structures for biological studies. Complicated chemical strategies in glycan synthesis are now more frequently replaced by regio- and stereo-specific enzymes. The exploration of microbial resources and improved production of mammalian enzymes have established glycosyltransferases as an efficient complementary tool for glycan synthesis. In this chapter, we demonstrate the feasibility of preparative enzymatic synthesis of different categories of glycans, such as blood group and tumor-associated poly-N-acetyllactosamines antigens, ganglio-oligosaccharides, N- and O-glycans. The enzymatic approach has generated over 100 novel oligosaccharides in amounts allowing milligram to gram distribution to many researchers in the field. Our diverse library has also formed the foundation for the successful developments of both the noncovalent enzyme-linked immunosorbent assay glycan array and the covalent printed glycan microarray.