A series of TiO2 nanotube arrays (TNAs) with different diameters and roughness were prepared by electrochemical anodization method by changing the fluoride ion concentration [0.4%, 0.3%, 0.2%(mass)] and applied voltage(15, 25, 35, 45 V). The scanning electron microscopy (FESEM) and atomic force microscopy (AFM) results showed that the wall thickness of the prepared TNAs increased and the roughness decreased with the decrease of fluoride ion concentration in the electrolyte. The effects of surface roughness and diameter on the surface mechanical properties of TNAs and the interaction of Cytochrome C (Cyt C) were studied by AFM characterization. The results show that the adhesion is proportional to the contact area. With the increase of the diameter of the TNAs, the wall thickness decreases, the effective contact area between TNAs and Cyt C increases first and then decreases, and the forces of Cyt C with TNAs also increase first and then decrease. The roughness decreases when the diameter was fixed, the effective area of TNAs increases, and the interaction force also increases. It can be seen that the surface roughness and effective contact area of TNAs can be effectively controlled by changing the fluoride ion concentration of the electrolyte, which is further beneficial to promoting interaction with protein molecules.