Fibroin from white silk has been widely investigated, particularly in the biomedical field. However, yellow silk, an abundant raw material source, has received much less attention. Therefore, this study focused on extracting fibroin from yellow silk (Bombyx mori L.) and formulating nanoparticles from the extracted fibroin, followed by evaluation of their physicochemical properties and biological characteristics. Fibroin was extracted from yellow silk through sericin removal and hot extraction, achieving an extraction yield of 79.21%. The nanoparticle system was prepared using the desolvation method, yielding particle sizes of 273-326 nm. The total nitrogen content of raw silk fibers, sericin-depleted fibers, fibroin, and fibroin nanoparticles, as determined by the Dumas method, ranged from 16.5% to 17%. Amino acid composition analysis of fibroin using HPLC showed that the major amino acids were glycine (43.7%), alanine (35.9%), serine (8.0%), and valine (2.8%). The surface charge, expressed as the point of zero charge (pH_pzc), of yellow silk, sericin-depleted silk fibers, and fibroin nanoparticles was 5.90, 5.95, and 5.45, respectively. Stability testing in strongly oxidative, strongly alkaline, and simulated gastrointestinal environments demonstrated that the fibroin nanoparticles maintained high stability. In addition, the system was non-irritating and showed no significant hemolytic toxicity. In conclusion, this study successfully extracted fibroin and formulated fibroin nanoparticles from yellow silk.