Titanium dioxide (TiO2) film is an important biomaterial used to improve the biocompatibility of titanium (Ti). We have used a film coating method with an aerosol beam and femtosecond laser irradiation to form periodic structures on biomaterials for control of the cell spreading. The control of cell spreading on biomaterials is important for the development of advanced biomaterials. In this study, nanostructures with periods of 130 and 230 nm were formed on a film using a femtosecond laser with wavelengths of 388 and 775 nm, respectively. The nanostructure period on the film was 30% of the laser wavelengths. Periods produced with wavelengths of 388 and 775 nm were calculated using a surface plasmon polariton (SPP) model and the experimental results for both wavelengths were in the range of the calculated periods, which suggests that the mechanism for the formation of the periodic nanostructures on the film with a femtosecond laser was due to the excitation of SPPs.