We synthesized fluorescent TEMPO derivatives which localize to membrane, mitochondria, or nuclei to evaluate oxidative stress in such biologically important organelles by using ESR spin probe technique. They bear fluorescein or Hoechst moiety as a fluorescent tag for confocal fluorescence microscopy. We introduced alkyl chain for targeting to membrane, or cationic moiety for targeting to mitochondria. For nuclear localization, Hoechst moiety was used as both fluorescence and a localizing function. However, Hoechst fluorescence is depending on its binding to DNA, so that it exhibits limited performance to ensure nuclear localization of the probe. So we report herein the synthesis and evaluation of a newly designed nuclear localizing TEMPO derivative with pyrrole polyamide, a well-known minor groove binder, as a localizing function and fluorescein as a dye.