Biochemical modeling of interaction between hyaluronidase and glycosaminoglycans (GAG) by their covalent attachment to enzyme had demonstrated that polymeric GAG (chondroitin sulfate, hyaluronan) contributed to stabilization of biocatalyst while copolymeric GAG (heparin, dermatan sulfate) inactivated the enzyme. These diversified effects are associated with structural distinctions of GAG following C-5 epimerization of glucuronic and iduronic acid residues, different effects of α[1—4] and α[1—3] compared to influence of β[1—4] and β[1—3] glycosidic linkages. Analysis of hyaluronidase glycosylation by di- and monosaccharides had shown significant influence of galactose C-4 epimers on endoglycosidase activity of enzyme compared to glucose, effective glycosylation of biocatalyst by disaccharide mixture (lactose, cellobiose, maltose) and importance of multiform microenvironment for enzyme function regulation. Elucidation of regulation mechanism for endoglycosidase activity of hyaluronidase required the new researching approaches with sequential study of conformational alterations of biocatalyst molecule during its interaction with GAG ligands.