A solvatochromic fluorophore, PRODAN, has been used as a microenvironment-sensitive reporter. Based on the chemistry of PRODAN, we designed and synthesized four novel fluorescent nucleosides, X-PDN (X = U, C, A, and G), to which a PRODAN fluorophore was attached at pyrimidine C5 or purine C8. The fluorescent nucleosides sensitively varied the Stokes shift values depending on the orientational polarizability of the solvent. The X-PDN incorporated into DNA also changed the Stokes shift values depending on the DNA structure. In particular, the excitation spectrum of the X-PDN-containing duplex shifted to a longer wavelength and gave a smaller Stokes shift value when the base opposite X-PDN could form a Watson-Crick base pair with X-PDN. A lower energy excitation of X-PDN-containing DNA resulted in a strong fluorescence emission selective to the Watson-Crick pairing base. This unique photochemical character was applicable to the efficient typing of single-nucleotide polymorphisms of genes.