Carbohydrates are biologically and medicinally important molecules that are attracting growing attention to their synthesis and applications. Unlike the biosynthetic processes for nucleic acids and proteins, carbohydrate biosynthesis is not template-driven, more challenging, and often leads to product variations. In lieu of templates for carbohydrate biosynthesis, we describe herein a new concept of designing enzyme assembly synthetic maps (EASyMaps) as blueprints to guide glycosyltransferase-dependent stepwise one-pot multienzyme (StOPMe) synthesis to systematically access structurally diverse carbohydrates in a target-oriented manner. The strategy is demonstrated for the construction of a comprehensive library of tetraose-core-containing human milk oligosaccharides (HMOs) presenting diverse functional important glycan epitopes shared by more complex HMOs. The tetraose-core-HMOs are attractive candidates for large-scale production and for the development of HMO-based nutraceuticals. To achieve the preparative-scale synthesis of targets containing a Neu5Acα2-6GlcNAc component, a human α2-6-sialyltransferase hST6GALNAC5 is successfully expressed in