Collagen porous scaffolds have been widely employed as a dermal equivalent to induce fibroblasts infiltration and dermal regeneration. To eliminate the disadvantageous drawback of the fast degradation speed, a cross-linking method was adopted by using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) and N-hydroxysuccinimide (NHS) in the presence of amino acids (glycin, glutamic acid or lysine), which function as cross-linking bridge between collagen molecular chains. In vitro assessment of the biological stability of the cross-linked collagen scaffolds found that the collagenase biodegradation degree was greatly decreased when lysine was added, resulting in a more biological stable scaffold. On the other hand, the biodegradation degree was accelerated compared with the purely cross-linked when glutamic acid was added, while less influenced by glycin addition. By comparing the biodegradation degree of the scaffolds added with amino acids and their model compounds, i.e. adipic acid and hexane diamine, the key factor influencing the biological stability was further investigated. The results indicated that the crucial factor is dependent on the ratio of amino groups to carboxyl groups in the cross-linking system. At optimal ratio the lowest biodegradation degree is achieved. Scanning electron microscopy measurements prove that the three-dimensional structure of the scaffolds was largely preserved. Preliminary in vitro culture of fibroblasts in the collagen scaffold cross-linked with EDAC/NHS in the presence of lysine has shown that the original good cytocompatibility of collagen was retained.