We designed and synthesized a nucleoside derivative in which the nucleobase is replaced with acridone. The nucleoside derivative was incorporated into an oligodeoxyribonucleotide (ODN), and its influence on the stability of ODN hybrids and its fluorescent properties in a DNA duplex were measured by thermodynamic analysis and fluorescent spectroscopy. These results showed that the acridonyl group could distinguish the type of nucleobase paired from fluorescent intensity, although the hybrid stability did not depend significantly on the types of nucleobases paired.