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In the last few decades, microspheres and microsphere-based scaffolds have demonstrated great potential in translational medicine. Various materials, including natural polymers, synthetic polymers, bioactive ceramics, and their composites, have been fabricated into microspheres for bone-defect repair. In this review, we summarize the specific steps and research progress of various fabrication methods for microspheres and identify the main limitations of existing techniques. The appropriate materials and surface-modification strategies that can increase the biological properties of microspheres are also described. Finally, we discuss the key application of microsphere-functionalized scaffolds for bone tissue engineering. We expect that this review will enable more future investigations that focus on the field of microsphere tissue engineering, and thus advance the development of fabrication technology and facilitate the application of microspheres for bone regeneration.