The invention relates to an ophthalmic in-situ jelly of chiral anti-glaucoma medicine levofloxacin tropane. The ophthalmic jellies concretely comprises 0.03 percent to 0.3 percent of levofloxacin tropane, 10 percent to 20 percent of hydrophilic polymer material, 0 percent to 20 percent of humectant, 0.1 percent to 10 percent of permeation pressure regulator, 0.01 percent to 0.3 percent of antiseptic/bacteriostat, pH value regulator and water. In a preparation method, the materials are weighted according to the percentage; the levofloxacin tropane is solved in injection water, the hydrophilic polymer material is added under the stirring state, and the solution is stood over a night; the adjuvant such as the humectant and the like is added, and the pH value is adjusted as 4 to 9; the solution is filtered, the injection water is added to the total quantity, and the ophthalmic in-situ jelly is obtained. The ophthalmic in-situ jelly has the advantages of uniform and smooth quality, proper viscosity, convenient use, exact dosage, and long-term uniform distribution on the surface of eye cornea; besides, the jelly causes no hypersusceptibility and irritation, no blurred vision, can effectively prolong the acting time of the medicine on the eye cornea, and has wide application prospect.