An in situ cross-linkable gelatin–poly(ethylene glycol)–tyramine (GPT) hydrogel was developed as a novel injectable material for tissue regenerative medicine. The hydrogel was formed rapidly using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The gelation times could be controlled from five seconds to over one minute by changing the HRP concentration, and the mechanical properties could be controlled by varying the H₂O₂ concentration. The storage moduli of the hydrogels ranged from approximately 200 to over 8000 Pa. In addition, an in vitrocell attachment study was carried out using various cell types. The results of this study demonstrated that the GPT hydrogel, formed in situ via an enzyme-mediated reaction, was an excellent bioactive matrix for cellular behavior. Furthermore, an in vitro 3D cell study was made using the GPT hydrogels formed with different mechanical strengths, and in vivo subcutaneous injection was carried out. The in vitro and in vivo studies demonstrated that the GPT hydrogels had excellent bioactivities, and that the cellular behavior in the hydrogel matrix could be controlled. The obtained results showed that these in situ forming GPT hydrogels, with excellent bioactivities and tunable physicochemical properties, have great potential for use as injectable materials in tissue regenerative medicine and various biomedical applications.