Because of their ease of handling and excellent biocompatibility, injectable macroporous hydrogels have received a considerable interest in the fields of tissue engineering and drug delivery systems because of their unique application in minimally invasive surgical procedures. In this study, in situ forming, injectable, macroporous, self-healing gelatin (GE)/oxidized alginate (OSA)/adipic acid dihydrazide (ADH) hydrogels were prepared using a high-speed shearing treatment and were stabilized by Schiff base reaction and acylhydrazone bonds. Their injectability, self-healing ability, rheology, microstructure, equilibrium water content, and in vitro biodegradation were investigated. We found that the injectable GE/OSA/ADH precursors remained in a liquid form and flowed easily for several minutes at room temperature, but however, gelled rapidly at body temperature. The gelation time could be regulated by varying the ratio of GE, OSA, and ADH. The obtained hydrogels had an interconnected macroporous structure and self-healing ability. The porosity of hydrogels was in the range of approximately 60–83%, and pore size varied from approximately 125–380 μm. The porous structure of hydrogel was visualized by field-emission scanning electron microscope, micro-computed tomography, and laser confocal microscope. Human epidermal growth factor was loaded by in situ mixing in GE/OSA/ADH hydrogels and was released with good bioactivity as evaluated by ELISA. Moreover, L929 cells proliferated on GE/OSA/ADH hydrogels, as verified by Cell Counting Kit-8 and LIVE/DEAD assays. Furthermore, encapsulation of NIH 3T3 cells within GE/OSA/ADH hydrogels demonstrated that the hydrogel can support cell survival, proliferation, and migration. In vivo studies showed that the hydrogels had a good injectability, in situ gelation, and tissue biocompatibility. Therefore, GE/OSA/ADH hydrogel represented a novel and safe injectable macroporous self-healing hydrogel for tissue engineering scaffold and drug delivery vehicle purposes.