Background: Conventional dosage forms of neomycin sulphate suffer from different drawbacks like spillage, poor penetration, low bioavailability, etc. In situ ophthalmic gel can overcome these problems by improving bioavailability, decreasing spillage and diminish the need for recurrent application. Thus, the aim of the present work was to prepare and evaluate in situ ophthalmic gel of neomycin sulphate for sustained ocular delivery.Method: Two polymers, sodium alginate (0.2-0.7%) and HPMC K4M, were used in different concentrations for the preparation of neomycin sulphate insitu gel. The prepared gel was subjected for rheological study, in vitro gelation, drug release study, antimicrobial study by disk diffusion method and others. Result: All formulations were found to be transparent and clear; pH of the formulations was within 6.8 to 6.9; drug content was found within 92-98% in all optimized in situ gelling systems. Viscosity of all formulations decreased as the shear rate increased, which indicated the character of pseudoplastic fluid. Optimized formulations F6 (0.5% Sodium alginate and HPMC K4M 1.5%), F7 (0.3% Sodium alginate and HPMC K4M 1.7%) and F8 (0.4% sodium alginate and HPMC K4M 2%) were liquid before instillation to the eye and underwent rapid gelation upon instillation to the eye. In vitro release through diffusion cell using cellophane membrane for 8 hours revealed sustained release of the drug from sodium alginate polymeric network over 6 hours. Study of antimicrobial efficacy using Pseudomonas aeruginosa for 24 hours showed all formulations to have effective antimicrobial action. Stability study for 6 months had revealed no changes in visual appearance, clarity, pH and % drug content. Conclusion: Hence, from the above results we can conclude that in situ ophthalmic gels of neomycin sulphate can be a good option for the treatment of various bacterial eye infections.