Abstract: Objective To develop a three-dimensional porous film of human hair keratin (HHK)-collagen sponge complex for use as a dermal substitute. Methods The three components F, B, and Z derived from healthy human hair were weaved into a meshwork and integrated with purified soluble type I collagen extracted from bovine tendons to prepare a highly porous film with vacuum freeze-drying followed by secondary cross-linking with glutaraldehyde. The film was grafted beneath the dorsal skin in 21 SD rats (experimental group), with simple collagen sponge serving as the negative control. The rats receiving surgical operation but without graft served as the blank control. The graft and its surrounding tissue were harvested on days 3, 7 and at weeks 2, 4, 6, 8, 12 after implantation for evaluation of tissue compatibility, vascularization and degradation. Results The prepared collagen sponge film was semitransparent and porous. Three to 7 days after grafting, inflammatory reaction was relieved gradually, and several fibroblasts and blood vessels were found adherent to the grafts in the experimental groups. At week 4, the wounds healed in the experimental groups, and the fibroblasts were actively secreting collagen and the film degraded obviously with the appearance of elastic fibers. At weeks 6 and 8, new collagen fibers thickened and assumed regular arrangement, and the collagen sponge films disappeared completely. In the control groups, the changes were less obvious and total HHK degradation occurred till week 12. Conclusion The degradable and absorbable HHK-collagen sponge film has relatively satisfactory tissue compatibility and can accelerate wound healing by stimulating cell proliferation and vascularization, showing the potential as an optimal dermal substitute.