The present investigation reports the formation and characterization of novel biocompatible chitosan nanoparticles based conjugated with fluorescent dye for biomedical applications. The solubility and size of these nanoparticles in the dried and swollen states will be described and discussed. The correlation of size of nanoparticles, pH of the solutions, ratio of cross-linking, and the dye-content of nanosystems have been studied. It was found, that biocompatible fluorescent chitosan nanosystems have been successfully prepared. The size and the size distributions of the nanosystems depend on the pH, but at a given pH, it was independent of the ratio of cross-linking. The diameter of individual dried particles varied in the range of 100-300 nm, and the average hydrodynamic diameters were in the range of 270-370 nm depending on the pH. The biocompatible fluorescent chitosan nanoparticles in aqueous media, might be useful for various in vitro experiments related to biomedical applications.