The purpose of this article is to develop an in-situ liquid crystal gel that can be used as a novel ocular delivery system for pilocarpine nitrate (PN). The phytantriol (PT) -based in situ liquid crystal gels were prepared by a vortex method using PT, PEG400, Triglyceride (TAG) and water (in the ratio of 61.15:30:3.85:5, w/w). Firstly, the internal structure of the PN-loaded liquid crystal gel was characterized by polarizing microscope (PLM), small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) and rheology. In vitro drug release behavior and ex vivo corneal permeation were investigated. Finally, eye irritation test，preocular residence time evaluation,were studied in vivo and compared with eye drops.Based on various characterization techniques, it is proved that the internal structure of the gel is a hexagonal phase.In vitro release results identified that PN could be released continuously from HII gel over a period of 24 h. The in vitro obvious permeability coefficient of HII gel was 3.19-fold (P < 0.01) higher than that of the eye drops. Compared with eye drops,the HII gel had good bioadhesion and displayed longer residence time on the eyeballs surface using fluorescent labeling technology.In addition, through Corneal hydration level and eye irritation test ., it is conjectured that HII gel will not cause eye irritation. In short, the formulation has the advantages of high efficiency, slow release and non-toxicity, and will become a promising pharmaceutical strategy to improve the efficacy of glaucoma.