<p>Silk fibroin has emerged as a promising natural biomaterial due to the exceptional biocompatibility, unique functional architecture, and controllable biodegradability. It can be processed into silk fibroin nanoparticles through multiple techniques, such as microfluidic. Owing to the unique structural features, the prepared silk fibroin nanoparticles exhibit favorable drug-loading capacities and intrinsic stimulus-responsive behaviors, which have enabled their broad application in advanced drug delivery systems. This review summarizes the extraction methods and structural features of silk fibroin, as well as the fabricating and modifying strategies, physicochemical properties, and applications of silk fibroin nanoparticles. We also summarize the major challenges associated with the large-scale production and clinical translation of silk fibroin nanoparticles, including limitations in manufacturing reproducibility and quality control. Finally, we discuss several potential strategies that may help to address these barriers, with the aim of narrowing-rather than fully resolving-the current gap between fundamental laboratory research and future clinical development.</p>