Inspired by the knowledge that one usual method to impart chirality to metal nanoparticles is to passivate them with ligands possessing chirality, in this context, a pair of optically active blue-emitting carbon dots (CDs) were prepared using citric acid and D/L-penicillamine (D/L-Pen) as the carbon sources through a two-step pyrolytic route. The second line of our research related to the effect of chiral CDs on cytotoxicity reveals that CDs coated with D-Pen, show less cytotoxicity than L-Pen-coated CDs, although generally the cytotoxicity of them is low. The subcellular localization examination reveals that the as-prepared CDs could specifically target and directly illuminate the Golgi apparatus, in spite that no obvious differences are observed between both of them. The discovery could provide a new alternative avenue to trace the cell state by real-time monitoring the Golgi morphology.