The morphology of self-assembled TiO2 nanotubes layer plays a key role in electrical conductivity and biocompatibility properties in terms of cell proliferation, adhesion and mineralization. Many research studies have been reported in using a TiO2 nanotubes for different medical applications, there is a lack of unified correlation between TNT morphology and its electrochemical properties. The aim of this study was to examine the effects of diameter and annealing conditions on TiO2 nanotubes with identical height and their behaviour as biosensor platform. TiO2 nanotubes layer, 1000 nm thick with nanotubes of diameters in range: 25 ÷ 100 nm, was prepared by anodizing of the titanium foil in ethylene glycol solution. To change the crystal structure and improve the electrical conductivity of the semiconductive TiO2 nanotubes layer the thermal treatment by annealing in argon, nitrogen or air was used. Basing on the electrochemical tests, the XPS and scanning microscopy examinations, as well as the contact angle measurements and the amperometric detection of potassium ferricyanide, it was concluded that the 1000 nm thick TiO2 nanotubes layer with nanotubes of 50 nm diameter, annealed in argon, showed the best physicochemical properties, which helps investigate the adsorption immobilization mechanism. The possibility of using TNT as a biosensor platform was confirmed in hydrogen detection.