Modifications at the glycerol side chain of sialic acid in sialosides modulate their recognition by sialic acid-binding proteins and sialidases. However, limited work has been focused on the synthesis and functional studies of sialosides with C7-modified sialic acids. Here we report chemical synthesis of C4-modified ManNAc and mannose and their application as sialic acid precursors in a highly efficient one-pot three-enzyme system for chemoenzymatic synthesis of α2-3- and α2-6-linked sialyl para-nitrophenyl galactosides in which the C7-hydroxyl group in sialic acid (N-acetylneuraminic acid, Neu5Ac, or 2-keto-3-deoxynonulosonic acid, Kdn) was systematically substituted by -F, -OMe, -H, and -N3 groups. Substrate specificity study of bacterial and human sialidases using the obtained sialoside library containing C7-modified sialic acids showed that sialosides containing C7-deoxy Neu5Ac were selective substrates for all bacterial sialidases tested but not for human NEU2. The information obtained from sialidase substrate specificity can be used to guide the design of new inhibitors that are selective against bacterial sialidases.