The development of controlled drug delivery systems for bone regeneration, especially microspheres, has become a research hotspot in recent years. Chitosan and its derivative O-carboxymethyl chitosan have been considered to be an effective way for controlled drug delivery due to their nontoxicity and biodegradability. Currently, most of the studies have researched on synthesizing and characterizing chitosan and O-carboxymethyl chitosan. However, few studies have focused on the differences between chitosan microspheres and O-carboxymethyl chitosan microspheres directly. In this study, chitosan and O-carboxymethyl chitosan microspheres were developed by water-in-oil emulsification cross-linking method using vanillin as the cross-linking agent, and then their physicochemical properties were evaluated by Fourier transform infrared spectroscopy, scanning electron microscopy, and in vitro release testing. The results showed that O-carboxymethyl chitosan was successfully modified by adding carboxymethyl group at the chitosan C6 position.The particle size of chitosan microspheres (50-90 mu m) was significantly larger than that of O-carboxymethyl chitosan microspheres (10-50 mu m), and the drug release profile of O-carboxymethyl chitosan microspheres showed larger initial burst release within the first day and sustained release at the fourth day, while chitosan microspheres showed sustained release at the seventh day. In addition, Cell Counting Kit-8 assay showed that MC3T3-E1 proliferated well and highly expressed the alkaline phosphatase marker protein on both chitosan and O-carboxymethyl chitosan microspheres. Overall, both chitosan and O-carboxymethyl chitosan microspheres showed good biocompatibility, and chitosan microspheres were superior to O-carboxymethyl chitosan microspheres. Moreover, the different drug release rates suggest that chitosan and O-carboxymethyl chitosan microspheres have the potential to be used for the repair of different bone defects.