This study aimed to optimize the cleaning process of the anodized specimens for fabricating clean NiTi-O nanotubes exhibiting visible light-mediated antibacterial activity at the surface of NiTi alloy. The cleaning process of the anodized specimens tested in this study was composed of two steps. The First step included two washing solvents (distilled water and heptane) with different washing times (1, 3, and 5 min) and temperatures (25 and 80 ℃), and the second step was treated by ultrasonicator (1, 3, and 5 min). From the results of FE-SEM observation, clean and uniform nanotubes (length: 600±80 nm, diameter: 52 ±5 nm) at the surface of NiTi alloy were observed on the condition of 5 min of washing in heptane at 25 ℃ and then 3 min of ultrasonication. Thin film XRD analysis resulted that the brookite TiO 2 crystal structure being detected in the anodized specimen. Furthermore, the live-dead assay resulted that there was no significant difference among the cell viability numbers of hMSCs cultured on a cell culture dish (control), NiTi alloy, and NiTi-O nanotubes (P>0.05). Therefore, the optimized cleaning process of the anodized NiTi alloy is expected to be more feasible for the NiTi alloy-based implant surface treatment technology.