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The aim of this study was to evaluate intraocular biodistribution of a fluorescent polymeric nanosystem composed of chitosan and gelatin after intravitreal administration in rat eyes. The nanoparticles based on chitosan and gelatin were synthesized using a reverse emulsion-double cross-linking technique (ionic and covalent) and their structural characteristics are presented. Two units of 1% suspension of fluorescein-labeled nanoparticles in saline solution were injected intravitreal in rat eyes. The histological cross-sections obtained at 24 and 72 hours were analyzed by confocal microscopy and compared to a similar number of control cross-sections. The scanning electron microscopy of the nanoparticles obtained by double cross-linking in reverse emulsion technique revealed spherical, smooth, highly porous particles with no tendency to form aggregates. The chitosan–fluorescein conjugate was present in all the ocular tissues both at 24 and at 72 hours. The nanoparticles were present in the retina in a larger quantity and persisted longer than in the other ocular tissues. They were mainly fixed paravascular. The double cross-linking in reverse emulsion technique was efficient in synthesizing a biocompatible polymeric nanosystem. The in vivo study of intraocular biodistribution of fluorescein-labeled nanoparticles revealed their affinity for the retina after intravitreal administration.