Chitosan microspheres with diameter of 14.3-48.5 μm were prepared by emulsion method and using natural vanillin as cross-linking agent. The surface morphology and microstructure of the microspheres were characterized by scanning electron microscopy, X-ray diffraction and Fourier-transform infrared spectroscopy, etc. The hollow microspheres showed a well-defined spherical shape with median diameter of 30.3 μm and possessed a uniform surface with micro-wrinkles, which is in favor of the drug release. Interpenetrating network cross-linking mechanism might result from the Schiff base reaction and the acetalization of hydroxyl and carbonyl between chitosan and vanillin. Berberine, as a model drug, was loaded in the microspheres and released in a sustainable manner. The drug loading ratio could change from 9.16% to 29.70% corresponding to the entrapment efficiency of 91.61% to 74.25%. In vitro cell culture study using MG63 cells and in vivo implantation clearly showed that the microspheres could provide favorable cell attachment and biocompatibility. The results confirm that the drug-loaded vanillin-crosslinked chitosan microspheres could be a worthy candidate either as carriers of drugs and cells, or as therapeutic matrix for bone repair and regeneration.