A series of novel 5′-labelled uridine with fluorophores: N-alkylcarbazole, alkyloxynaphthalene and alkylfluorene were synthesized through H-phosphonate by Arbuzov reaction, and were characterized by using ¹H NMR, ³¹P NMR and ESI-MS analysis. The fluorescence spectra of all the compounds in solution and solid powder were described. The experimental results showed that the fluorescence emission intensity of 5′-labelled uridine is very sensitive to solvents. Copyright © 2010 John Wiley & Sons, Ltd.