Abstract: Chemical method was adapted to prepare the key substrate , l – α – phosphatidylinositol(PI）, of Listeria monocytogenes(LM） chromogenic medium. The purity and the yield were contradict factors which could not be improved both in the extraction of PI. The experiment combined single and mixed solvent methods to extract PI, especially investigated the influences of solvent ratio, solvent volume, the kind of alkalinity substances to the purity and yield of the products. The optimal conditions appeared after pretreatment of alkali methanol, under the solvent ratio of V(n-hexane) ： V(ethanol solution) = 1:1.5, using aqueous ammonia as alkalinity substance, and pH = 8.0. The comparison of color development among the PI before and after the purification and the product purchased from abroad, showed that the product had a promising application future.