Abstract: The application of Silk Fibroin (SF) nanospheres for controlled-release drug delivery was investigated. Drug-loaded SF nanospheres were rapidly prepared by nanoprecipitation method from SF and drug aqueous solution by using acetone as a non-solvent. Gentamicin sulfate was used as a model water-soluble drug. The drug-loaded nanospheres with 160-180 nm in size were spherical in shape and had a smooth surface. The SF nanosphere matrices were changed from random coil to β-sheet conformation by acetone treatment and drug entrapment. FTIR spectroscopy and thermogravimetric analyses indicated intermolecular interactions between SF and drug molecules. The cumulative in vitro drug release profiles from SF nanospheres showed initial rapid burst release followed with sustained-release behaviors. The drug release rate increased with the decreasing drug ratio. All these findings suggest that the SF nanospheres are potentially useful in drug delivery systems, especially hydrophilic drugs.