Objective To determine the differences in mechanical properties of TiO2 nano-film prepared by the method of anodic oxidation on different titanium alloy surfaces (TA1, TC4, TC4F136) under the same processing conditions. Methods SEM and AFM were used to observe morphology and structure of these three kinds of samples. The UNHT nanoindentor was used to test the mechanical property of TiO2 nano-film, and nano scratch was performed to determine the adhesive strength and the friction property between TiO2 nano-films on the three titanium alloy surfaces and the substrate. Nanoindentation was used to test microhardness and elastic modulus of TiO2 nano-films. Results It showed that the structure (tube diameter, wall thickness and length) of TiO2 films formed on different titanium alloy substrate differed from each other although they were prepared in the same electrolyte and other electrochemical conditions. Conclusion The mechanical properties of TiO2 nano-films on titanium alloy substrates which are prepared by anodic oxidation depend on the micromorphology of TiO2 and its adhesive strength with the substrates which ultimately depend on the difference of elementary composition of those alloys.