The drawbacks of poloxamer hydrogel, such as dissolving quickly in aqueous solution, have limited its biomedical application. In order to improve the stability of hydrogel, a novel system was developed by combining the reversible thermo-sensitive property of poloxamer 407 and the thiol-ene reactivity between the acrylate and thiol groups. It was found that the sol-gel transition of the acrylate/thiol modified poloxamer 407 mixture could be achieved at body temperature even with a low concentration of 17.5 wt.%. Meanwhile, the reaction between the acrylate and thiol modified poloxamer 407s occurred spontaneously in mimic physiological conditions, thus the hydrogel with crosslinking structure was formed. As a result, the stability of the crosslinked hydrogel was enhanced remarkably, and the release time of the drug from the crosslinked hydrogel was about 4.0 times as long as that from the poloxamer 407 hydrogel. Invitro and invivo experiments revealed that the biocompatibilities of the modified poloxamer 407 hydrogel were similar to that of poloxamer 407. These results indicate that the modified poloxamer 407s have potential applications in controlled drug release, tissue engineering and cell encapsulation etc.