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The objective of the current investigation is the development and characterization of in-situ ophthalmic formulation of fluconazole for sustained release at the eye site for a prolong period of time. Sol-gel fluconazole in-situ formulation was prepared by thermo-responsive pluronic F127, Na+ activated sodium alginate alone and in combination, characterization parameters include FTIR spectroscopy, Visual assessment and clarity test, Gelling ability test, pH testing, drug assay, In-vitro drug release and optimized formulation was evaluated for In-vitro antifungal studies by comparing with marketed formulation of fluconazole. Optimized formulation F8 composed of fluconazole (0.3%w/v), Pluronic F127(1%w/v), sodium alginate (0.5%w/v), sodium chloride (0.9w/v), benzalkonium Chloride (0.01%w/v) and acetate buffer pH 4 up to 100%w/v), showed drug-polymer compatibility as per FTIR, high gelling consistency on contact with simulated physiological conditions, 6.9 pH, drug assay was estimated (99.89±0.78 %) with 100% in-vitro drug released for about 6 hours. The in-vitro antifungal study was found to be 18.87±0.65 mm; 20.76±0.23 mm in comparison to marketed fluconazole conventional gel formulation (15.98±0.98 mm; 18.98±0.76 mm) for Candida albicans and Aspergillus niger pathogenic fungal strains respectively. Prepared in-situ ophthalmic sol-gel formulation of fluconazole could be considered as efficient delivery system to sustained the drug at the target site and effectively eradicating deeply rooted pathogenic fungal stains. [ABSTRACT FROM AUTHOR]