Abstract: The size reference material is used to test and calibrate apparatus, evaluate measure method, and ensure sample values. It is important in industry measurement, controling of products quality and science studying. In the present paper, crosslinked polystyrene microspheres with an average size of about 3.0 m were prepared by dispersion polymerization. Compared with the micro PS particle prepared from linear PS, the PS micro particle prepared from cross-linked PS shows superior resistance to solvent and heat, with the glass transition temperature increasement of 28.6 ℃ and remains insoluble in normal solvents. F-test and t-test were used to test the uniformity and stability of the sample. The quantification was carried out by independent measurement techniques named scanning electron microscope (SEM), which was calibrated with NIST Standard Reference Material (SRM2800). It is shown that the sizes of these PS spheres samples are 2.84 m and the expanded uncertainty is 0.08 m. The quantification process, the uniformity and the stability of these samples meet the demands of secondary standard reference materials of China.