We report a novel fluorescent nucleobase, (DNC)U, for the mapping of the local dielectric properties around DNA. Fluorescence spectra of the nucleoside d(DNC)U showed a significant shift depending on the solvent polarity. This property was used to determine the dielectric constants of the interior of DNA-binding proteins, such as DNA polymerases. We were able to demonstrate that the DNA-binding region of the Klenow fragment is a lower polarity site, as shown by the blue shift of the fluorescence peak originating from (DNC)U.