The aim of the present research work was to formulate, optimize and evaluate the in-situ gel for the ophthalmic drug delivery using the combination of gellan gum and carbopol 934P. The Box-Behnken design was applied to optimize the concentration of gellan gum (X), carbopol 934P (X) and benzododecenium bromide (X) to achieve the maximum viscosity [at physiological condition; 35 °C, pH 7.4, and simulated tear fluid (STF)], mucoadhesive strength, permeability coefficient and sustained release of the drug from the gel with constraint on the viscosity under the non-physiological condition (25 °C, pH 5). Response surface plots were drawn, the statistical validity of the polynomials was established, and optimized formulation was selected by the feasibility and grid search. The design proposed the optimized batch by selecting the independent variables as gellan gum (0.55% w/v), carbopol 934P (0.35% w/v) and benzododecenium bromide (0.013% w/v) to achieve the maximum viscosity (3363 cps) at physiological condition, mucoadhesive strength (22.35 dyn/cm), t (1200 min), permeability coefficient (1.36 × 10 sq.cm/sec), with minimum viscosity (131 cps) under the non-physiological condition. The combination of gellan gum and carbopol 934P improved the gelation (synergistic effect) characteristics of the in situ gel. The optimized in situ gel was clear, isotonic, pH 4.7 and showed pseudoplastic flow, high in vitro gelling capacity, low contact angle, acceptable hardness (51018 gm), compressibility (64617 gm) and adhesiveness (74 gm) values for the ocular application. The ex vivo study showed the significant protection of the mast cell from the degranulation. The ocular irritation and histopathology studies in the rabbit eyes confirmed the safety of in situ gel for human use. The in vivo drug release studies showed the presence of drug in the rabbit tear fluid up to 3 h in comparison to just 1 h with the eye drop solution. The contact time of the in situ gel in the human eye was 15.0 ± 2.5 min, which was >2 folds higher than the marketed gel (6.0 ± 3.2 min), which could reduce the dosing frequency and total dose of drug. The Box-Behnken design facilitated the optimization of in situ gel for sustained ophthalmic drug delivery.