A versatile hydrogel was developed for enhancing bioactive wound healing by introducing the amphiphilic GHK peptide (GHK-C16) into a photo-crosslinkable tyramine-modified hyaluronic acid (HA-Ty). GHK-C16 self-assembled into GHK nanofibers (GHK NF) in HA-Ty solution, which underwent in situ gelation after the wound area was filled with precursor solution. Blue light irradiation (460-490 nm), with riboflavin phosphate as a photoinitiator, was used to trigger crosslinking, which enhanced the stability of the highly degradable hyaluronic acid and enabled sustained release of the nanostructured GHK derivatives. The hydrogels provided a microenvironment that promoted the proliferation of dermal fibroblasts and the activation of cytokines, leading to reduced inflammation and increased collagen expression during wound healing. The complexation of Cu into GHK nanofibers resulted in superior wound healing capabilities compared with non-lipidated GHK peptide with a comparable level of growth factor (EGF). Additionally, nanostructured Cu-GHK improved angiogenesis through vascular endothelial growth factor (VEGF) activation, which exerted a synergistic therapeutic effect. Furthermore, in vivo wound healing experiments revealed that the Cu-GHK NF/HA-Ty hydrogel accelerated wound healing through densely packed remodeled collagen in the dermis and promoting the growth of denser fibroblasts. HA-Ty hydrogels incorporating GHK NF also possessed improved mechanical properties and a faster wound healing rate, making them suitable for advanced bioactive wound healing applications. STATEMENT OF SIGNIFICANCE: By combining photo-crosslinkable tyramine-modified hyaluronic acid with self-assembled Cu-GHK-C16 peptide nanofibers (Cu-GHK NF), the Cu-GHK NF/HA-Ty hydrogel offers remarkable advantages over conventional non-structured Cu-GHK for wound healing. It enhances cell proliferation, migration, and collagen remodeling-critical factors in tissue regeneration. The incorporation of GHK nanofibers complexed with copper ions imparts potent anti-inflammatory effects, promoting cytokine activation and angiogenesis during wound healing. The Cu-GHK NF/hydrogel's unique properties, including in situ photo-crosslinking, ensure high customization and potency in tissue regeneration, providing a cost-effective alternative to growth factors. In vivo experiments further validate its efficacy, demonstrating significant wound closure, collagen remodeling, and increased fibroblast density. Overall, the Cu-GHK NF/HA-Ty hydrogel represents an advanced therapeutic option for wound healing applications.