A novel method to prepare crosslinked polyethyleneimine (CPEI) hollow nanospheres was reported. Uniform silica nanospheres were used as templates, 3-aminopropyl trimethoxysilane (APS) was immobilized on the surface of silica nanospheres as couple agent. Aziridine was initiated ring-opening polymerization with the amino groups in APS to form polyethyleneimine (PEI) shell layer. 1,4-Butanediol diacrylate was utilized to crosslink PEI polymeric shell. The silica nanospheres in core were etched by hydrofluoric acid to obtain hollow CPEI nanospheres. The hollow nanospheres were characterized by X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), and thermogravimetric analysis (TGA).