Titanium alloy scaffolds have recently gained substantial interest for the treatment of critical-size bone defect, particularly along with the maturity of the 3D printing technology that is capable of turning scaffold design ideas into real implants. As titanium alloys lack surface osteogenic activity, for improved biological performance of such scaffolds, surface modification is necessary. Various coating materials and coating methods have been explored. In this study, we developed a unique surface modification method to provide the surface of 3D printed titanium scaffolds with nano-sized structure and bioactive agent. Uniform, ordered TiO₂ nanotube arrays were formed by applying two-step anodization, and then mesoporous bioactive glass (MBG) was loaded into nanotubes. The results of in vitro immersion testing showed that bioactive ions, i.e., Si and Ca ions, could be steadily and continuously released from MBG into basal medium. The assessment of the responses of hBMSCs confirmed that the surface-modified scaffolds supported the adhesion and proliferation of hBMSCs, indicating good surface cytocompatibility. The developed method of combining surface nanostructure and bioactive agent could be used as a new strategy to improve the osteogenic activity of 3D printed titanium alloy scaffolds.