Gelatin is an important hydrocolloid which has widespread applications in the pharmaceutical industry and biomedical science. However, despite its significance, clinical applications of gelatin are largely limited because of its gel-to-solution transition around human body temperature. In this work, we developed a series of F127-gelatin composite hydrogels with reverse thermo-responsive properties. The stiffness of F127-gelatin hydrogels can be adjusted by varying the types and contents of gelatin (type A, GA and type B, GB). The results of rheological data showed that F127-GA gels have higher gel strengths than those of F127-GB, which might be due to different isoelectric points between GA and GB. In addition, F127-gelatin gels showed significant cell viability and biocompatibility for human mesenchymal stem cells, indicating that these discovered hydrogels can serve as potential biomaterials and implantable scaffolds for applications in tissue engineering and regenerative medicine.