•The developed latanoprost-loaded hydrogel showed a sustained release profile.•The developed hydrogel showed the both in-vitro and in-vivo biocompatibility.•Intraocular pressure was decreased within 7 days after treatment with the hydrogel.•Latanoprost acid was detected in the aqueous humor for 7 days.•The hydrogel may provide a way for long-term intraocular pressure control. Ocular hypertension is a major risk factor for the development and progression of glaucoma. Frequent and long-term application of latanoprost often causes undesirable local side effects, which are a major cause of therapeutic failure due to loss of persistence in using this glaucoma medical therapy. In the present study, we developed a thermosensitive chitosan-based hydrogel as a topical eye drop formulation for the sustained release of latanoprost to control ocular hypertension. The developed formulation without preservatives may improve compliance and possibly even efficacy. The results of this study support its biocompatibility and sustained-release profile both in vitro and in vivo. After topical application of latanoprost-loaded hydrogel, triamcinolone acetonide-induced elevated intraocular pressure was significantly decreased within 7 days and remained at a normal level for the following 21 days in rabbit eyes. This newly developed chitosan-based hydrogel may provide a non-invasive alternative to traditional anti-glaucoma eye drops for glaucoma treatment.