This study was aimed to prepare, characterize and evaluate in situ gel formulations based on a blend of chitosan (CS), polyvinyl alcohol (PVA) and gellan gum (Gelrite™) for a sustained ocular delivery of besifloxacin (BSF). The developed formulations were evaluated for physicochemical properties, gelation time (Tsol-gel), rheological behaviour, antimicrobial efficacy, pharmacokinetic assessment, gamma scintigraphy study and ocular irritation. The results showed BSF sol-gel system were found to be sensitive enough which underwent instantaneous phase transition upon getting physiological stimulation. The ex vivo permeation experiments indicated that the developed formulation was able to enhance the retention of BSF at corneal surface. The HET-CAM confirmed the non-irritancy of developed formulation and also demonstrated the ability of ocular protection against strongly irritant substances. The results of gamma scintigraphy study revealed the higher concentration of drug retains at the corneal surface. In addition, optimized BSF sol-gel system showed enhanced anti-bacterial activity compared to BSF suspension.