Acellular Dermal Matrix (ADM) is mainly made with human or porcine skins and has the risk of zoonotic virus transmission. The fish skin-derived ADM could overcome the shortcoming. Fish skin acellular matrix has been used as wound dressing, but there is few systematic studies on tilapia-skin acellular dermal matrix (TS-ADM). In the present study, a novel TS-ADM was made by an alkaline decellularization process and γ-irradiation. The physical properties, biocompatibility, pre-clinical safety and wound healing activity of TS-ADM were systematically evaluated for its value as a functionally bioactive wound dressing. Histopathological analysis (hematoxylin and eosin staining, 4,6-diamidino-2-phenylindole (DAPI) staining) and DNA quantification both proved that the nuclear components of tilapia skin were removed sufficiently in TS-ADM. Compared to the commercial porcine acellular dermal matrix (DC-ADM), TS-ADM has distinctive features in morphology, thermal stability, degradability and water vapor transmission. TS-ADM was more readily degradable than DC-ADM in vitro and in vivo. In both rat and mini-pig skin wound healing experiments, TS-ADM was shown to significantly promote granulation growth, collagen deposition, angiogenesis and re-epithelialization, which may be attributed to the high expression of transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA) and CD31. Herein, the novel TS-ADM, used as a low-cost bioactive dressing, could form a microenvironment conducive to wound healing. A novel wound dressing, tilapia skin acellular dermal matrix (TS-ADM) made with a low-cost process, promotes wound healing via extracellular matrix deposition, angiogenesis and re-epithelialization. The partially degraded TS-ADM may form a microenvironment that enhances the expression of transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA) and CD31. • Tilapia skin was processed into a novel TS-ADM by a low-cost process that enables efficient decellularization. • TS-ADM was more readily resorbable relative to a commercial porcine acellular dermal matrix in vitro and in vivo. • TS-ADM could promote granulation growth, angiogenesis and re-epithelialization and improve the quality of regenerated skin.