Glatiramer acetate (GA), a commonly used treatment for multiple sclerosis (MS), requires long-term frequent injections to ensure its effectiveness. This often leads to adverse effects, patient noncompliance, and economic inefficiency. In this study, poloxamer, as a thermosensitive polymer modified by chitosan (CS) and hyaluronic acid (HA), was employed to prepare an in situ forming prolonged release formulation of GA to overcome the problems derived from frequent repeated injections and to enhance the patient compliance. The sol-gel formulation was produced through a cold method and optimized using design of experiments. The final product was characterized in terms of gelation time (GT), rheological behaviors, morphological properties, assay, and drug release kinetics. The in vitro release rate of GA during the first 24 h was quite rapid, but then it continued at a slower rate of 0.05 mg ml-1h-1. The in vivo analysis after the subcutaneous injections showed lower levels of IL-5, IL-13, and uric acid (UA) in mice treated with the gel formulation compared with those receiving free GA in the first few days. However, after 10 days, significantly higher concentrations were detected, which continued to increase slowly. It can be concluded that the designed thermosensitive sol-gel formula is capable of extending the effectiveness of GA and can be considered as a promising sustained release formulation for the treatment of MS.