Using the transglycosylation reaction as a reverse reaction for the hydrolysis of hyaluronidase, new artificial oligosaccharides may be synthesized by reconstructing natural glycosaminoglycans (GAGs) according to preliminary planned arrangements. However, as some problems have been associated with the method, including the low yields of reaction products and complicated processes of separation and purification, improvements in this method were investigated. Transglycosylation reactions were carried out using bovine testicular hyaluronidase-immobilized resin packed in a column. For the transglycosylation reaction, pyridylaminated (PA) GAG hexasaccharides, which were the minimum size for hydrolysis sensitivity and the transglycosylation reaction, were used as acceptors, whereas large size GAGs were used as donors. The reaction mixture was pooled after incubation in the hyaluronidase-immobilized resin column and was then introduced into continuously joined HPLC columns constructed from three steps: the first step of ion-exchange HPLC for concentrating newly synthesized GAG oligosaccharides as reaction products, the second step of reverse phase HPLC for separating PA oligosaccharides from non-PA oligosaccharides, and the third step of size fractionation HPLC for fractionating newly synthesized oligosaccharides. Newly synthesized oligosaccharides were obtained by one complete cycle of the transglycosylation reaction and separation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 189–196, 2014.