Silk fibroin (SF) is a natural polymeric material that has attracted intense research attention in the field of wound healing due to its exceptional mechanical properties, tunable biodegradability, and multifunctional bioactivity. This review systematically summarizes the preparation strategies, functional modifications, and multidimensional application mechanisms of SF and its composite materials in wound healing. The innovative applications of SF in intelligent dressing design, immunometabolic regulation, controlled drug release, stem-cell function modulation, and bioelectrical-activity-mediated microenvironment remodeling is further explored, while analyzing the therapeutic efficacy and cost-effectiveness of SF through clinical translation cases. Distinct from previous reviews, this work not only integrates the latest advances in SF molecular mechanisms and material design but also emphasizes its potential in precision medicine, such as the development of genetically engineered SF for customized immunoregulatory networks. Finally, the article highlights the current challenges in the development of SF materials, including mechanical stability, degradation controllability, and standardization of large-scale production, and envisions future research directions driven by 3D bioprinting and synthetic biology technologies. This review provides a theoretical foundation and technical reference information for the development of efficient, multifunctional, and clinically translatable SF-based materials for application in wound healing.