Objective To explore the biological safety and compatibility of five kinds of acellular fish skin matrices, and to screen the materials meeting the criteria of biocompatibility. Methods Acellular fish skin matrices were prepared through decolorization, degreasing, decellularization and cross-linking using five kinds of fish skins leather jacket fish (Thamnaconus septentrionalis), black fish (Channa argus), eel (Anguillidae), carp (Cyprinus carpio) and silver crap (Hypophthalmichthys molitrix). According to the national standard (GB/T 16886.5-2017), different concentrations (10 g/L and 100 g/L) of extracts of acellular fish skin matrices were prepared, and the CCK-8 assay was used to detect the in vitro toxicity of extracts of acellular fish skin matrices to human bone marrow-derived mesenchymal stem cells. The surface structures of different collagen membranes were observed under scanning electron microscopy. The H-E and Masson staining were used to observe whether the fish skin matrix materials were completely acellular. The human bone marrow-derived mesenchymal stem cells were cultured with the medium containing standard concentration of 100 g/L acellular fish skin matrix material extracts. The blood compatibility of the meterial was tested by hemolysis test. Live/Dead, TUNEL and flow cytometry were used to detect the cell survival and apoptosis levels. Results CCK-8 assay showed that the viability indexes of the cells co-cultured with the leather jacket fish and black fish skin acellular matrix material extracts were both >70% at the concentration of 10 g/L, and the other three materials were all <70%. At the concentration of 100 g/L, the viability index of the cells co-cultured with the leather jacket fish skin acellular matrix material extracts was >70%, and that with the black fish was <70%. Scanning electron microscopy showed that the leather jacket fish skin acellular matrix materials had porous and compact structure inside, and black fish skin acellular matrix materials had smooth structure inside. The H-E staining and Masson staining showed that leather jacket fish and black fish skin acellular matrix materials were both acellular collagen fibers. The hemolysis rates (the national standard ≤ 5%) of leather jacket fish and black fish skin acellular matrix material extracts were (1.23±0.43)% and (6.35±0.47)%, respectively. Human bone marrow-derived mesenchymal stem cells survived well in the culture medium containing extracts of leather jacket fish skin acellular matrix material, but most cells died or apoptozed in the culture medium containing extracts of Black fish skin acellular matrix material. Conclusion The leather jacket fish decellularized skin matrix material has better biosafety and biocompatibility, and has the potential to be a scaffold material for tissue engineering.