Glycosyltransferases are important synthetic enzymes for the construction of naturally occurring glycoconjugates as well as for the design of neoglycoconjugates. The assay methods currently available for these enzymes require tedious and time-consuming procedures for separation of products and do not permit continual assay of enzyme activities. As a set of convenient fluorogenic substrates for continuous monitoring of sialyltransferase activities, we designed and synthesized a novel CMP-Neu5Ac derivative with a naphthylmethyl group at the C-9 position and Nacetyllactosamine derivative containing a dansyl group at the terminal position of aglycon. In such substrates, the emission peak of the naphthylmethyl group ( em โซุโฌ 340 nm) of the glycosyl donor is successfully overlapped with the excitation peak due to the dansyl group ( ex โซุโฌ 335 nm) of the glycosyl acceptor. A coupling reaction of these two substrates catalyzed by rat liver 2,6-sialyltransferase caused an increase of dansyl fluorescence ( em โซุโฌ 525 nm) and a decrease of naphthylmethyl fluorescence on the basis of resonance energy transfer between two fluorescence probes. The substrates presented here permit continuous fluorescent monitoring of enzymatic sugar combining reactions. Actually, using this time course of enzymatic reactions, kinetic constants of rat liver 2,6sialyltransferase against glycosyl donor substrates were estimated to be K m โซุโฌ 4.85 M and V max โซุโฌ 0.119 mol/min, respectively. This strategy allows precise and efficient analyses of enzyme kinetics not possible with the conventional assay methods for the glycosyl-transferases that usually require separation of products from the reaction mixture.