Highly photoluminescent C-Dots are prepared by hydrothermal reaction of citric acid and piperazine as dual reason in both hydrolysis and surface passivation agent along with the N-doping agent. The resulting C-Dots without external passivation showed PL enhancement by electron transfer from C-Dots-donor to piperazine-acceptor with the maximum emission yields of more than 84%. After mixing the prepared C-Dots with the mixture of EtOH and acetone (1:1 vol %), a novel washing process was performed by centrifugation to remove the precursors and by-products. The resulting C-Dots showed much better stability than dialysis. For evaluating the possible application of the C-Dots as a fluorescent biological probe which possess the wide dosing window and multicolor properties. The C-Dots were localized in perinuclear vacuole-like structures with granular pattern in cytoplasm sparing the nucleus. No signs of any morphological deterioration like nuclear shrinkage were observed.