Titanium (Ti) is widely used for biomaterials such as an implant for human body due to its excellent anti-corrosion. However, new functions should be added to Ti substrates because a strong revival is slow in a joint with a bone. A useful method is periodic nano structure formation on a material to control cell spreading and promote the osteogenic differentiation of cells. When a femtosecond laser is irradiated on a Ti plate at a fluence of near the ablation threshold, periodic nano structures are formed in the direction of perpendicular to the polarization angle of the laser pulse. In our previous study, it was found that the direction of cell was spread by periodic nano structures formed on Ti plate. For the controllability improvement of the cell spreading direction, it is required to improve the uniformity of periodic nano structures. Since the period of nano structures depend on the target material, irradiation fluence, number of irradiation-pulses, incidence angle, and laser wavelength, it is difficult to control the uniformity. In this study, to improve the uniformity of nano structures, Ti plate was irradiated by two-color double-pulse irradiation with a femtosecond laser at the wavelength of 800 nm. As the results, when the 800 nm and 400 nm pulses had orthogonal polarization directions, high uniformity of periodic nano structure was formed.