Collagen, as a major structural protein in the extracellular matrix, plays a crucial role in tissue regeneration; however, traditional collagen sources often suffer from immunogenicity, poor stability, and batch-to-batch variability. In this study, a novel transdermal peptide-recombinant type III collagen hydrogel was developed, which has excellent biocompatibility, thermal stability, and skin repair effects. Transdermal peptide-recombinant type III collagen was expressed and purified by genetic engineering methods, and its potential for biomedical applications was further evaluated. The experimental results showed that the prepared collagen had a high purity (95%) and retained the unique secondary structure of collagen, showing good structural stability at different pH values. Through cell proliferation experiments and mouse wound healing experiments, we verified the superior effect of the collagen hydrogel in promoting wound healing and significantly accelerating wound healing at concentrations as low as 0.2 mg/mL. This achievement provides strong experimental support for its application in clinical skin wound repair.