A platform for sequential glycosylation reactions (SUGAR-TARGET) was developed to enable precise, controlled N-linked glycosylation of proteins. This platform uses immobilized enzymes to perform targeted glycosylation in vitro, allowing for the synthesis of homogeneous glycan structures. The system involves the immobilization of glycosyltransferases through a one-step biotinylation and purification method, enabling spatiotemporal separation of reactions to minimize enzyme competition and achieve high glycan homogeneity. The platform was tested with various glycosyltransferases, including N-acetylglucosaminyltransferase I (GnTI), α-mannosidase II (ManII), β-1,4-galactosyltransferase (GalT), and β-galactoside α-2,6-sialyltransferase I (SiaT), to produce homogeneous glycoforms. The system was applied to modify the galactosylation profile of immunoglobulin G (IgG) antibodies, resulting in increased terminal galactosylation. Immobilized GalT was shown to be reusable for over 80 hours, demonstrating the platform's potential for industrial applications. The study highlights the ability of SUGAR-TARGET to produce homogeneous glycoprotein structures, which is crucial for the development of therapeutics with defined glycosylation profiles. The platform offers a modular and scalable approach to glycoengineering, enabling the synthesis of tailored glycoforms for functional and clinical evaluation.A platform for sequential glycosylation reactions (SUGAR-TARGET) was developed to enable precise, controlled N-linked glycosylation of proteins. This platform uses immobilized enzymes to perform targeted glycosylation in vitro, allowing for the synthesis of homogeneous glycan structures. The system involves the immobilization of glycosyltransferases through a one-step biotinylation and purification method, enabling spatiotemporal separation of reactions to minimize enzyme competition and achieve high glycan homogeneity. The platform was tested with various glycosyltransferases, including N-acetylglucosaminyltransferase I (GnTI), α-mannosidase II (ManII), β-1,4-galactosyltransferase (GalT), and β-galactoside α-2,6-sialyltransferase I (SiaT), to produce homogeneous glycoforms. The system was applied to modify the galactosylation profile of immunoglobulin G (IgG) antibodies, resulting in increased terminal galactosylation. Immobilized GalT was shown to be reusable for over 80 hours, demonstrating the platform's potential for industrial applications. The study highlights the ability of SUGAR-TARGET to produce homogeneous glycoprotein structures, which is crucial for the development of therapeutics with defined glycosylation profiles. The platform offers a modular and scalable approach to glycoengineering, enabling the synthesis of tailored glycoforms for functional and clinical evaluation.