Abstract: In order to solve the problems of the poor stability and high hemolytic toxicity of Temporin-GHaR, the preparation design technology was used to construct a thermosensitive quaternary composite hydrogel of carboxymethyl chitosan/hydroxypropyl methylcellulose/poloxamer loaded with Temporin-GHaR (CHPP/GHaR). The response surface method was used to optimize the formulation, and the obtained hydrogel with a porous fiber network structure exhibited shear-thinning, self-healing, and injectability, with a swelling rate of 161.5%. The release experiments tests showed a sustained release of 55% within 40 hours with low burst release risk. Biocompatibility evaluation test confirmed that the hydrogel loaded with 200 μmol·L⁻¹ GHaR had only 2.2% hemolysis rate and no significant cytotoxicity to keratinocytes and umbilical vein endothelial cells. In vitro antimicrobial experiments demonstrated that CHPP/GHaR achieved above 99% inhibition rates against methicillin-resistant Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Our findings provide a new candidate material for the treatment of the drug-resistant bacterial-infections.