Acellular matrix is a type of promising biomaterial for wound healing promotion. Although acellular bovine and porcine tissues have proven effective, religious restrictions and risks of disease transmission remain barriers to their clinical use. Acellular fish skin (AFS), given its similarity to human skin structure and without the aforementioned disadvantages, is thus seen as an attractive alternative. This study aims to fabricate AFS from the skin of black carp (Mylopharyngodon piceus), evaluate its physical and mechanical properties and assess its impact on wound healing. The results showed that AFS has a highly porous structure, along with high levels of hydrophilicity, water-absorption property and permeability. Furthermore, physical characterization showed the high tensile strength of AFS in dry and wet states, and high stitch tear resistance, indicating great potential in clinical applications. Cell Counting Kit-8 was used to test the viability of L929 cells when culturing in the extracts of AFS. Compared with the control group, there is no significant difference in optical density value when culturing in the extracts of AFS at days 1, 3 and 7 (*p > 0.05). In vivo wound healing evaluation then highlighted its promotion of angiogenesis and collagen synthesis, its function in anti-inflammation and acceleration in wound healing. Therefore, this study suggests that AFS has potential as a promising alternative to mammal-derived or traditional wound dressing.