Biomaterials are widely used in regenerative medicine to repair full-thickness skin defect wounds. The adipose-derived stromal vascular fraction (SVF) shows pro-regenerative properties, however, the ex vivo biological activity of SVF is suppressed due to the lack of an external scaffold. Tilapia skin, as a sustained and recyclable biomaterial with low immunogenicity, was applied in the preparation of a hydrogel. The mixture of tilapia skin-derived gelatin and methacrylic anhydride as a scaffold facilitated the paracrine function of SVF and exerted a synergistic effect with SVF to promote wound healing. In this study, 30% (w/v) SVF was added to methacrylate-functionalized tilapia skin gelatin and subsequently exposed to UV irradiation to form a three-dimensional nano-scaffolding composite hydrogel (FG-SVF-3). The effects of paracrine growth factors, neovascularization, and collagen production on wound healing were extensively discussed. FG-SVF-3 displayed a pronounced wound healing ability via in vivo wound models. The FG-SVF-3 hydrogel enhanced the biocompatibility and the expression of EGF, bFGF, and VEGF. FG-SVF-3, as a promising wound dressing, exhibited superior ability to accelerate wound healing, skin regeneration, and wound closure.