Objective To manufacture fish swim bladder membrane material by crosslinking techniques, and to explore its physical and chemical properties and cytotoxicity. Methods After decellularization, the swim bladders were randomly divided into two groups. The swim bladders were treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) crosslinking method, surface hole making, and freeze-drying in crosslinking group, and only surface hole making and freeze-drying in non-crosslinking group. The physical and chemical properties of the materials were observed, including microstructure by scanning electron microscopy (SEM), mechanical properties (tensile strength and breaking elongation) by universal tensile machine, hydrophilicity by contact angle measuring instrument, porosity by ethanol infiltration method, degradation performance in vitro and thermal stability test, and the components of materials by infrared spectrum analysis. Mouse fibroblasts (L929) were cultured with the extracts of two groups of materials in order to determine the cytotoxicity of materials by using cell counting kit 8 (CCK-8) method. Results The porous structure and rough surface of materials were observed by SEM. Compared with the non-crosslinking group, the tensile stress of the crosslinking group was higher, the breaking elongation was lower, and the porosity increased, showing significant differences (P<0.05). There was no significant difference in contact angle between the two groups (P>0.05). The degradation was faster within the first 7 days and then tended to be smooth in the two groups. But the degradation rates of crosslinking group were significantly lower than those of non-crosslinking group (P<0.05). Differential scanning calorimeter showed that the denaturation temperature of the crosslinking group was (75.2±1.3)℃, which was significantly higher than that of the non-crosslinking group [(68.5±0.4)℃] (t=4.586, P=0.002). Compared with the non-crosslinking group, the crosslinking group produced new C=O bond and N-H bond, and no other new groups were introduced into the cross-linking group. CCK-8 method showed that the absorbance values of the crosslinking group and the non-crosslinking group were not significant when compared with the positive control group (P>0.05). Conclusion The fish swim bladder membrane obtained by crosslinking treatment with EDC/NHS method has good physical and chemical properties, no cytotoxicity, and is expected to be used as a dura mater repair material.