AbstractSialyloligosaccharides of the type Neu5 Acα2‐3Galβ1‐3GalNAc and a range of corresponding motifs play an important role in nature and are found in gangliosides, Lewis type I structures and the sialyl‐Thomsen–Friedenreich antigen occurring in higher animals, viruses, bacteria, protozoa and pathogenic fungi. There is considerable interest to evaluate the significant functionalities of these glycostructures, and here we present a chemoenzymatic approach by a facile synthesis of such motifs. Employing chemoenzymatic methods, several modified Galβ1‐3GalNAc derivatives were synthesized. Modifications were introduced at the stage of the monomeric building blocks prior to formation of the disaccharides by means of four different β‐galactosidases from bovine testes, Bacillus circulans and Xanthomonas manihotis as well as the phosphorylase from Bifidobacterium bifidum. Finally, the modified disaccharide derivatives could be efficiently sialylated using the recombinant trans‐sialidase from Trypanosoma cruzi.