A thermally reversible nanogel is used in capillary electrophoresis to create discrete regions for a galactosyltransferase reaction and separation. The β1-4 galactosyltransferase enzyme, donor, and co-factor were patterned in the capillary. The substrate was driven through these zones and converted to galactosylated products, which were separated and identified. Using this capillary electrophoresis method, the degree of glycosylation was discernible for a pentasaccharide and for biantennary N-glycans. With the ability to distinguish between reaction products for which either one or two galactose residues were transferred, the capillary nanogel electrophoresis system was used to determine the Michaelis-Menten value, KM. For the β1-4 galactosyltransferase, the KM value obtained for a pentasaccharide substrate was 1.23 ± 0.08 mM. Once KM was established, the enzyme/substrate ratio was evaluated to add a single galactose residue or to fully galactosylate a biantennary N-glycan. Additionally, capillary nanogel electrophoresis was adapted to transfer galactose residues to protein. The applicability of the method for real-time online modification of whole protein was demonstrated with the Herceptin glycoprotein. Complete retardation by Erythrina cristagalli lectin after enzymatic modification confirmed the addition of galactose residues to the Herceptin. This demonstrated the potential of the method to be used for online modification of other glycoproteins.