PicrosideⅡ, one of the most effective components extracted from picrorhiza, has been reported with neuronal protective activity. To isolate the picroside Ⅱ and similar structure moleculars from picrorhiza effectively, molecularly imprinted polymer (MIP) was prepared by precipitation polymerization with Picroside Ⅱ as template molecule and TRIM as cross-linkers. The MIP was with high affinity and excellent stereo-selectivity toward Picroside Ⅱ and similar structure molecular. The purity of picroside is 70% of target sample. This research might offer a novel method for the enrichment and determination of target compounds from the Traditional Chinese Medicine. A model of middle cerebral artery occlusion and reperfusion (MCAO/R) in rats was used to determine the protective effects of Picroside II sample of this experiment on cerebral ischaemia/reperfusion in vivo. Results indicate the picroside II sample can protect neurons against acute ischemic stroke injury, and the neurological deficit scores of dosage group is lower than that of model group.