Biological matrices can be modified with cross‐linkers to improve some of their characteristics as scaffolds for tissue engineering. In this study, chemical cross‐linker 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC) was used with different ratios (5, 10, 20, 30, and 40 mM) to improve properties such as mechanical strength, denaturation temperature, and degradability of the acellular fish skin as a biological scaffold for tissue engineering applications. Morphological analysis showed that the use of cross‐linker at low concentrations had no effect on the structure and textiles of the scaffold, while increasing mechanical strength, denaturation temperature, and degradation time. Cytotoxicity and cellular studies showed that the optimal cross‐linker concentration did not significantly affect cell viability as well as cell adhesion. In general, utilising the carbodiimide cross‐linker with the optimal ratio can improve the characteristics and function of the biological tissues such as acellular fish skin.