Immobilization of a thyroid hormone substitute of L-thyroxine on chitosan-based smart hydrogel structure was performed to reduce the side effects of the required daily dose. The hydrogel was obtained by the Schiff reaction of chitosan with acetaldehyde and quaternization with methyl iodide then by ion exchange with NaCl salt. Finally, a hydrogel was obtained from the treatment of chitosan-based salt at -200C. The amount of levothyroxine immobilized by physical sorption on the hydrogel was determined by molecular electron spectroscopy on basis especially absorption peak at 227 nm. Results of IR- and UV-Vis spectroscopic analyses revealed that the interaction between levothyroxine and hydrogel occurs mainly due to hydrogen bonding and orientation forces of intermolecular interaction