Objective:To isolate and prepare the reference substances of phloroglucinol derivatives from Dryopteris fragrans by semi-preparation HPLC. Methods:After reflux extraction of Dryopteris fragrans with petroleum ether,the extracting solution was con-centrated and separated by silica gel column chromatography,and then isolated by semi-preparation HPLC. The isocratic elution was carried out using acetonitrile as the mobile phase at 3. 0 ml·min-1 and the injection volume was 0. 5 ml. Two phloroglucinol deriva-tives were isolated. Results:The chemical structure of the two phloroglucinol derivatives respectively was aspidin BB with the purity of 98. 81% and aspidin PB with the purity of 98. 57% by ultra high performance liquid chromatography. Conclusion:The isolation of as-pidin BB and aspidin PB by semi-preparation HPLC is simple and fast with the purity over 98%,which can be used to prepare the ref-erence substances.