The cell-free filtrate of S. coelicoflavus was used as a reducing agent and a capping agent for the biosynthesis of low-toxic, high-stable and biocompatible silver nanoparticles (AgNPs). The morphology, crystal structure, size distribution, elemental composition and surface-capping groups of biosynthesized AgNPs were characterized. Moreover, the biochemical mechanism for the extracellular synthesis of AgNPs was also explored by analyzing the important roles of the bioactive molecules secreted by S. coelicoflavus during the biosynthesis process. The results demonstrated that the prepared AgNPs presented in spherical or approximately spherical shape followed by a small amount of truncated triangular, quadrangular and hexagonal nanoplates with an average particle size of 34.50 nm, mid-range polydisperse, with good crystallinity and polycrystalline structure. Proteins and reduced glutathione (GSH) secreted by S. coelicoflavus were responsible for the process of AgNPs biosynthesis. Transmembrane protein and bacterioferritin were capped on the surface of the prepared AgNPs to maintain the high stability of AgNPs in the reaction system. Nitrate reductase was likely to co-catalyze the reduction of silver ions with other bioactive components.