The development of reliable, eco-friendly processes for the synthesis of nanomaterials is an important aspect of nanotechnology today. One approach that shows immense potential is based on the in situ synthesis of noble metal nanocolloids using natural biological material such as silk fibroin. In the present paper, the nano-Ag/silk-fibroin colloids were prepared by in situ reducing AgNO3 with silk-fibroin at room temperature without any reducing agent. UV-Vis absorption, atomic force microscopy, fluorescence spectra and resonance scattering spectra were used to study the preparation process and characterization of the nanocomposite. It was found that the formative Ag nanoparticles were attributed to the tyrosine residues in the chains of silk-fibroin that act as reduction agent in the reduction course of AgNO3, and the Ag nanoparticles were uniformly embedded in silk-fibroin colloid, which possess good dispersity and stability and can be kept for a long period. The strength of fluorescence spectra increased after reaction of silk-fibroin with AgNO3 solution, showing that there was a strong chemical adsorption between silver nanoparticles and silk-fibroin, and a stable complex layer was formed in silver nanoparticles. Meanwhile the existence of silver nanoparticles in silk-fibroin was proved by resonance scattering spectra.