Porous hollow silica spheres were synthesized using silk fibroin spheres as novel single sacrificial template via a sol-gel route and their in vitro biocompatibility and drug delivery property were evaluated. TEM observation showed that silica spheres had a hollow cavity and a porous shell, while BET measurement showed that they had the pore size of 22 nm and specific surface area of 45 m2/g. FT-IR spectrum showed that silica spheres contained SiOSi bonds. After exposure to Hela cells, MTT assay indicated that silica spheres were in vitro biocompatible. It was found that silica spheres not only supported adsorption of tetracycline hydrochloride (TH, one of the representative antibiotics) with adsorption efficiency of 17.4%, but also exhibited a sustained release behavior for TH. The TH-loaded silica spheres showed antibacterial property to inhibit the growth of two representative bacteria: Escherichia coli and Staphylococcus aureus, indicating that the released TH was biologically active.