Fabrication and characterization of TiO₂ nanotube arrays on Ti6Al4V surface for enhancement of cell treatment in biomedical applications was the aim of this study. In this regard, the anodizing process has been carried out in an organic bath (ethylene glycol + 0.5 wt% NH₄F + 3 wt% water) under applied voltages of 25 to 100 V. The anodized samples were characterized using scanning electron microscopy (SEM), hydrophilic/hydrophobic balance, cell attachment, ion release test and MTT assay. Based on achieved results, the nanotube arrays with good uniformity, high wettability and viability can successfully formed on Ti6Al4V surface in voltage range of about 50 to 75 V. The dimensional characteristics of formed nanotubes depend on applied voltage and play an essential role in viability and cell adhesion. In this work, the formed nanotubes array in applied voltage of 60 V (with average diameter and thickness of about 90–97 and 20–30, respectively) showed the best wettability, viability and cell response in comparison with other voltages.