A new Cu(I) probe (OBEP-CS1), bearing an alkyl-pyridinium moiety to get complete water solubility, was synthesized. Fluorescence titrations support copper binding and high selectivity in a water solution whereas formation of aggregates was excluded by DOSY experiments. The detection capability for intracellular Cu(I), especially localized in mitochondria, was demonstrated in differentiated neuroblastoma cells by confocal microscopy.