Colloidal lithography is one of the developing lithography techniques for the precision pattern definition without the limitation of optical diffraction. This study developed the fabrication, self-assembly arrangement and etching of polystyrene micro/nano spheres. The emulsion polymerization and dispersion polymerization were introduced to prepare nano, sub-micron and micro particle of polystyrene spheres (PS), respectively. Then using of the self-assembly arrangement to build the hexagonal close-packed sub-micron and micron spheres on the silicon substrate. Finally, three sizes of PS spheres, 450 nm, 550 nm and 700 nm, were used as etching masks in two-step etching process approach to fabricate periodical subwavelength pyramidal structure.