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Traumatic brain injury (TBI) is one of the most devastating nervous injuries. Neural tissue engineering based on stem cells and bioactive scaffold is a promising but challenging approach for neural repair. A cutting-edge system with capability to control the fate of encapsulated stem cells is attractive to enhance neural regeneration after TBI. Herein, an injectable gelatin hydrogel dual-enzymatically cross-linked by horse radish peroxidase (HRP) and choline oxidase (ChOx) was performed as the neural scaffold to load murine bone marrow-derived mesenchymal stem cells (BMSC) for TBI treatment. The results of in vitro cellular experiments showed that low cross-linked gelatin hydrogel could obviously promote cellular viability, neural differentiation, and neurotrophins secretion of the loaded BMSC. In vivo tests on a TBI model of C57BL/6 mouse demonstrated that BMSC-laden gelatin hydrogel implants could significantly reduce the damaged area, ameliorate inflammation, attenuate neuronal apoptosis, facilitate survival and proliferation of endogenous neural cells, and promote the neurological function recovery of TBI mice. All data suggest that establishment of this three-dimensional (3D) gelatin hydrogel stem cell-loaded system is a promising therapeutic strategy for TBI or other neurological rehabilitation. [Display omitted] • An injectable and dual-enzymatically cross-linked gelatin hydrogel was used to load BMSC for TBI treatment • Low cross-linked gelatin hydrogel obviously enhances cellular viability, neural differentiation, and neurotrophin secretion • This GH/MSC hydrogel implantation could promote the neurogenesis and functional reconstruction of TBI mice [ABSTRACT FROM AUTHOR]