Human skin substitutes are needed for implantation and wound repair based on the new concept of tissue engineering in combination with biomaterials and cell biological technology. However, failure sometimes occurs when the wound healing is delayed in vivo due to acute inflammation resulting from the early degradation of the transplanted biomaterials. Accordingly, the current study modified conventional biomaterials to overcome early degradation and strong inflammation. In a conventional skin substitute, the animal origin collagenous materials have a slight antigenicity as xenogenic materials, however, the modified method was able to obtain a low antigenicity and anti-inflammation effect using atelo-collagen and an amniotic component. The tyrosine content in the developed atelo-collagen, representing the antigenicity, was reduced from 0.590% to 0.046% based on an HPLC analysis. In addition, to reduce the inflammation and foreign material reaction, an amniotic component was applied to the atelo-collagen materials. While, to enhance the wound healing, the modified skin substitute was developed as a composite matrix of an atelo-collagen scaffold with an amniotic membrane component. A quantitative analysis of hEGF in the amniotic membrane was also performed using different processing methods. Finally, a tissueengineered skin substitute was constructed by cultivating skin cells in the collagen scaffold attached to an amniotic membrane.