In the study, bioactive apatite formation on the plasma electrolytic oxidation (PEO)-treated Ti-6Al-4V alloy after 3rd anodic titanium oxidation (ATO) was researched. After formation of the nanotubes on Ti-6Al-4V alloy in NaF solution, the nanotubes layer was removed via ultra-sonication, and the ATO was then repeated. After ATO treatment, the PEO-treatment was carried out in the electrolyte containing bioactive substances. The morphology and composition on Ti-6Al-4V was observed by field-emission scanning electron microscopy and energy dispersive X-ray spectrometer. The PEO film formed on the Ti-6Al-4V alloy was characterized using AFM, XRD, nanoindentation test, wettability test, and scratch test. The diameter and size of the nanotubes increased while the distance between the nanotubes decreased with increases in the number of ATO cycles. A mesh-shaped surface was formed and the surface roughness increased with the number of ATO cycles. Mn precipitates were formed around the pores, and Mg was more distributed on the surface than in the pores. The PEO-treated surface after 3rd ATO mainly exhibited anatase and hydroxyapatite phases. The PEO-treated surface exhibited higher bonding strength and the surface roughness increased with the number of ATO cycles. The wettability, the elastic modulus, and hardness increased as the number of ATO cycle increased in the number of ATO cycles.