Biodegradable chitosan hollow nanospheres were fabricated by employing uniform poly-D,L-lactide-poly(ethylene glycol) (PELA) nanoparticles as templates. Chitosan was adsorbed onto the surface of PELA nanoparticle templates through the electrostatic interaction between the sulphuric acid groups from sodium dodecyl sulfate (SDS) on the templates and the amino groups of the chitosan. Subsequently, the core-coated structure of chitosan-PELA nanospheres was obtained with the adsorbed chitosan layer being further crosslinked with glutaraldehyde. After the removal of the templates, PELA cores, chitosan hollow nanospheres were achieved. The mean size and size distribution of these nanospheres were measured with dynamic light scattering. The hollow structure was identified by transmission electron microscopy, atomic force microscopy and laser confocal scanning microscope. The antitumor drug model, adriamycin hydrochloride, was adsorbed on/into the chitosan hollow nanospheres. The drug release behaviors were investigated in phosphate buffered solution (PBS) at pH 7.4 and acetate buffered solution (ABS) at pH 4.5, respectively, at 37°C, and in vitro tumor cell growth inhibition assay was also evaluated. The biodegradable hollow nanospheres possess great potential applications in nanomedicine.