The invention discloses a thermo-sensitive hydrogel. The thermo-sensitive hydrogel comprises the following raw materials: a cytokine activity stabilizer, an epidermal stem cell conditioned culture medium and a collagen cross-linking solution. The cytokine activity stabilizer is added into the epidermal stem cell conditioned culture medium, so that multiple cytokines secreted by EpSCs are stabilized, the half-life period of each of the multiple cytokines is prolonged, the effect of each of the multiple cytokines on trauma repair is prolonged, and further, wound healing is promoted. In addition, the invention discloses a preparation method of the thermo-sensitive hydrogel, which has the characteristics of being simple to operate, random in molding and capable of in-situ gelling; the prepared hydrogel is slightly-viscous and clear liquid under the condition of 4-25 DEG C, is a solid gel when being put under the condition of 30-37 DEG C, has better mechanical strength and better biological compatibility and can promote fibroblast proliferation and facilitate fresh skin apparent and functional recovery, so that wounds are healed with high quality.