The objective of this study was to develop an ion-activated in situ gelling vehicle for ophthalmic delivery of matrine. The rheological properties of polymer solutions, including Gelrite, alginate, and Gelrite/alginate solution, were evaluated. In addition, the effect of formulation characteristics on in vitro release and in vivo precorneal drug kinetic of matrine was investigated. It was found that the optimum concentration of Gelrite solution for the in situ gel-forming delivery systems was 0.3% (w/w) and that for alginate solution was 1.4% (w/w). The mixture of 0.2% Gelrite and 0.6% alginate solutions showed a significant enhancement in gel strength at physiological condition. On the basis of the in vitro results, the Gelrite formulations of matrine-containing alginate released the drug most slowly. For each tested polymer solution, the concentration of matrine in the precorneal area was higher than that of matrine-containing simulated tear fluid (STF) almost at each time point (p < 0.05). The area under the curve of formulation 16 (0.2%Gelrite/0.6%alginate) was 4.65 times greater than that of containing matrine STF. Both the in vitro release and in vivo pharmacological studies indicated that the Gelrite/alginate solution had the better ability to retain drug than the Gelrite or alginate solutions alone. The tested formulation was found to be almost non-irritant in the ocular irritancy test. The overall results of this study revealed that the Gelrite/alginate mixture can be used as an in situ gelling vehicle to enhance ocular retention.