Green-fluorescent biocompatible carbon dots with a quantum yield of 40% were successfully synthesized through a solvothermal process and then they are comprehensively characterized. The carbon dots showed a negatively charged surface owing to the presence of carboxylic groups. This negative surface charge hinders the effective targeting and imaging of mitochondria. To address this limitation, a new approach is developed in this study. An amphiphile containing phenylalanine, with a positively charged polar head consisting of triphenylphosphine and a hydrophobic aliphatic tail, was designed, synthesized, purified, and characterized. This amphiphile formed spherical micelle-type nanostructures in an aqueous medium in the aggregated state. Although these nanoprobes lack inherent fluorescence, they exhibited the capability to image mitochondria when their spherical micelle-type nanostructures were decorated with negatively charged fluorescent nanocarbon dots in both cancerous (KB cells) and non-cancerous (CHO cells) cell lines. Notably, carbon dots without the amphiphile failed to penetrate the cell membrane as they exhibited significantly low emission inside the cell. This study extensively explored the cell entry mechanism of the hybrid nanoprobes. The photophysical changes and the interaction between the negatively charged carbon dots and the positively charged nanospheres of the amphiphile were also analyzed in this study.