The methylated DNA base 5-methylcytosine (C-m) is the most important factor in the epigenetic regulation of gene expression. 3-cyanovinylcarbazole nucleoside (CNVK) photocrosslinks with C-m more rapidly than with cytosine (C). In the present study, we evaluated the effect of the substituents of various 3-vinylcarbazole derivatives on C-m- and C-mediated DNA photocrosslinking for the quantitative detection of C-m. We also demonstrated the quantitative detection of C-m via photocrosslinking using 3-vinylcarbazole derivatives on a DNA chip. The results suggest that this photocrosslinking reaction has potential for the selective detection of C-m in DNA strands via photocrosslinking using 3-cyanovinylcarbazole derivatives.