Ofloxacin is a synthetic broad-spectrum fluroquinolone antibiotic, frequently used for the treatment of ocular infections. In the present study, ionic gelation technique was employed as the method of preparation of ofloxacin loaded chitosan nanoparticles using different concentrations of chitosan and sodium tripolyphosphate (TPP) and the potential of these nanoparticles in sustained ocular drug delivery was investigated. Different drug-polymer ratios were used to prepare five batches of nanoparticles at two concentrations of chitosan solutions (0.2% w/v and 0.4% w/v). The formulated nanoparticles were evaluated for morphology by Scanning Electron Microscopy, and subjected to particle size analysis and zeta potential measurement by Zetasizer. Drug content, encapsulation efficiency and in vitro drug release studies were also done. LCS-OF3 nanoparticles prepared with 0.2% chitosan solution and drug-chitosan ratio 3:1 were checked for pH and osmolality for their suitability in in vivo study after that subjected to in vivo drug release study in rabbits, the results of which clearly indicate the controlled and prolonged drug release from LCS-OF3 nanoparticles when compared to that from commercially available ofloxacin eye drops.