3D printed medical titanium alloy has shown great advantages in manufacturing complex and personalized scaffolds for tissue engineering. To promote the repair of bone defects, fabrication of a scaffold with high accuracy and excellent cell behaviors is required. However, the partially melted or un-melted powders must be removed in the post-process. In this study, the effects of unfused metal powders on the biocompatibility of 3D printed Ti alloy scaffolds were analyzed by co-culture with MC3T3-E1 cells and RAW 264.7 cells, and the surface treatment strategy was proposed. Results showed that surface treatment can enhance the biocompatibility of the 3D printed Ti alloy scaffolds. The vitro tests with MC3T3-E1 cells demonstrated that the ETi5Cu (etched Ti5Cu) showed the better cell proliferation and osteogenic potential, and the lower cell apoptosis rates. Moreover, higher concentration of powders showed inhibitory effects on cytocompatibility and osteogenesis of MC3T3-E1 cells, and promoted the polarization of macrophages to M1 type. In summary, the removal of metal powders on the 3D printed scaffolds plays an important role in the biological performance, which provided a promising strategy for the application of Ti implants.