Abstract: OBJECTIVE To prepare parishin B reference substance for quantitative analysis. METHODS Parishin B was isolated and purified by macroporous resin column chromatography, silica gel column chromatography, and preparative HPLC. Its structure was identified by UV, IR, ESI-MS and NMR spectral techniques. The purity was determined by HPLC and TLC. Finally, the stability was investigated under different temperature and illumination. RESULTS The reference substance of parishin B was prepared for Gastrodia elata and its purity was >99.0% in line with the standard of quantitative analysis. CONCLUSION Parishin B prepared by this method can be used as reference substance to the quality control for natural product and their preparations, as well as the research of pharmacodynamic material basis.