The Bombyx mori silk fibroin microspheres with controllable size were prepared by electrostatic spraying and freeze-drying method. The effects of solution concentration, voltage and flow rate on the sphere size were discussed. The morphology of microspheres was observed by scanning electron microscopy (SEM). The influence of ethanol treatment on the molecular conformation of silk fibroin microspheres was investigated by XRD and FT-IR spectra. The results indicated that the silk fibroin microspheres with diameter in range of 117-363 μm were spherical in shape, and there were plenty of pores both on the surface and in the interior of the microspheres. The sphere size increased with the rising of silk fibroin solution concentration and flow rate, while decreased as the voltage enhanced. The conformation of silk fibroin microspheres changed from random coil to silk II structure after ethanol treatment.