Micron-level porous rough surface was firstly constructed on the surface of titanium alloy by 3D-printing technology, and then nano-level structure was constructed on the micron-level porous rough surface by surface technology. Firstly, the surface of 3D-printed titanium alloy was pretreated by the combination of high pressure water treatment and acid etching treatment to remove the spherical powder particles with bad bonding and reduce the surface roughness and anisotropy of 3D-printed titanium alloy. Then, through anodic oxidation treatment, TiO2 periosteum layer with nanoscale holes was prepared on the surface of 3D-printed titanium alloy. This preparation method solves the problems of large anisotropy on the surface of 3D printed titanium alloy, which leads to uneven electric field discharge in the subsequent electrochemical process and poor stability of TiO2-like periosteum layer formation. The occurrence of unstable discharge in anodic oxidation treatment is inhibited, the surface color of the film is uniform, and it is firmly combined with titanium or titanium alloy. Thus, the nano-layer structure of 3D printed titanium alloy micro-surface is constructed. As a result, the film formation rate and stability of 3D-printed titanium alloy surface film prepared by this process are good, it‘s a three-dimensional network, there are small holes in the big hole, the structure is conducive to cell adhesion, differentiation and proliferation on its surface, and is an ideal medical implant material.