Background: Collagen and chondroitin sulfate (CS) are an essential component of the natural extracellular matrix (ECM) of most tissues. They provide the mechanical stability to cone the compressive forces in ECM. In tissue engineering, electrospun nanofibrous scaffolds prepared by electrospinning technique have emerged as a suitable candidate to imitate natural ECM functions. Cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride/N-hydroxy succinimide can overcome the weak mechanical integrity of the engineered scaffolds in addition to the increased degradation stability under physiological conditions. Materials and methods: This study has synthesized nanofibrous collagen-CS scaffolds by using the electrospinning method. Results: The results have shown that incorporation of CS in higher concentration, along with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride/N-hydroxy succinimide, enhanced mechanical stability. Scaffolds showed more resistance to collagenase digestion. Fabricated scaffolds showed biocompatibility in corneal epithelial cell attachment. Conclusion: These results demonstrate that cross-linked electrospun CO-CS mats exhibited a uniform nanofibrous and porous structure, especially for lower concentration of the cross-linker and may be utilized as an alternative effective substrate in tissue engineering.