In this work, water-soluble Ag nanoparticles were prepared in aqueous solution by using trypsin as reducing and capping agent. The protein-assisted synthetic strategy eliminates the need of intermediate protecting and linking agents compared with organometallic approach, which is simple, effect, less energy consuming, and closer to the requirements of green chemistry. The morphology, size and antibacterial activity properties could be controlled by varying experimental conditions. The results of FT-IR and SDS-PAGE analysis indicated that trypsin molecules could control the nucleation and growth of nanocrystals through chemical interaction between Ag and functional groups of trypsin. The binding of trypsin on the surface of Ag nanoparticles significantly reduced nano toxicity through capping effect. The trypsin-conjugated Ag nanoparticles exhibited strong antibacterial activity toward both Gram-positive and Gram-negative bacteria due to small size and specific morphologies. Compared with traditional antibacterial materials, the water-solubility and biocompatibility make the products more suitable for the application in biological and medical science.