To clarify the cis-trans isomeric effect on the ultrafast DNA photo-cross-linking reaction of 3-cyanovinylcarbazole nucleoside (K-CNV) in the DNA duplex, which gives a single photodimer on the reversed-phase HPLC chromatogram, the kinetics of the cis-trans photoisomerization of K-CNV in double-stranded DNA was evaluated. Since the photoisomerization rate constant for cis to trans isomerization in double-stranded DNA was significantly larger than that for trans to cis isomerization, and the thermodynamic stability of the trans isomer was higher than that of the cis isomer, it was strongly suggested that the trans isomer of K-CNV is a reactive species of the photo-cross-linking reaction. H-1-H-1 NOESY analysis of the photoadduct consisting of K-CNV and T also supported the trans-mediated photo-cross-linking reaction of K-CNV. By using this ultrafast photo-cross-linking reaction for the molecular beacon-based SNPs typing, four individual Japanese rice strains were clearly distinguishable with simple photoirradiation and fluorescence imaging using double-stranded target DNAs.