Surface microstructure of biomaterials plays an important role in osteogenesis. In this study, we prepared a series of hydroxyapatite (HA) scaffolds (HA0, HA10, HA30, HA50) by 3D printing microspheres with different particle sizes (<60 μm). These scaffolds have similar physical and chemical properties, but form different microstructures due to their different particle size of microspheres, which result in certain impact on their biological properties. Compared with the traditional scaffolds without micro-sphere structure (HA0), the microsphere-based hydroxyapatite scaffolds provide improved adhesion and growth sites for bone mesenchymal stem cells (BMSCs) after 24 h culture, among which HA30 scaffold significantly promotes the pseudopodia elongation of BMSCs. After 5 d of cell culture on the scaffolds, the micro-sphere based HA scaffolds significantly stimulate proliferation of the BMSCs in contrast to the HA0 group. HA30 scaffolds with microsphere at similar size to the cell size have the best promoting effect on cell proliferation. Therefore, 3D printing technology can not only control the macrostructure of HA scaffolds, but also construct the microstructure on the surface of HA scaffolds by controlling the particle sizes of bioceramic powders to optimize the biological effects, which shows great potential application in the field of bone tissue engineering.