BACKGROUND: Current oral restoration membranes are expensive with a potential risk of zoonotic virus, so new membranes are in need. OBJECTIVE: To prepare fish skin acellular dermal matrix and evaluate its efficiency of decellularization, biomechanical properties and biocompatibility, in order to explore its feasibility as an oral restoration membrane. METHODS: The skin of Astroconger Myriaster was harvested to prepare acellular dermal matrix by decellularization via a combination of physical method and chemical and biological reagents. The micromorphology of the material was observed under scanning electron microscope. Tissue composition was analyzed by hematoxylin-eosin, Masson and PAS staining. The DNA content was detected using a kit. The biocompatibility was evaluated through cytotoxicity and animal skin stimulation tests. RESULTS AND CONCLUSION: The prepared acellular dermal matrix: (1) was white or light yellow, with strength and toughness; (2) exhibited a double-layered structure composed of a dense layer and a loose layer; (3) was mostly composed of collagen without PAS positive substance, showing good decellularization; (4) had a significantly lower DNA content than natural fish skin and commercial oral restoration membrane; and (5) possessed good biocompatibility, with very low cytotoxicity and no stimulation to the animal skin. Therefore, the prepared acellular dermal matrix of fish skin shows a potential in the preparation of oral guided tissue regeneration membrane.