3,6'-Disinapoylsucrose (DISS) is one of the main bioactive components in Polygalae Radix with known pharmacological effects. We herein developed a new column chromatographic extraction (CCE) method for the extraction of DISS from Polygalae Radix and a preparation method for further separation and purification. Polygalae Radix powder was loaded into a column with 1.5-fold volume (v/w) of 70% ethanol and soaked for 4 h. The column was then eluted with 70% ethanol at room temperature, only the first three fractions (each with 1.5-fold volume of the material weight) were collected as extracts and other fractions were sequentially circulated to the next column. The extraction rate of cyclic CCE procedure exceeded 99.4% which was 14.7% higher than the reflux extraction (extracted twice with 8-fold volume of 70% ethanol). The extracts were further purified by reversed-phase flash chromatography (RP-FC). The parameters such as mobile phase composition, sample scale and flow rate were investigated and optimized. The extracts were loaded onto a C-18 silica gel column with a sample/C-18 silica gel ratio of 1/10 (w/w). The column was eluted with ethanol/acetonitrile/water (11.25/3.75/85, v/v/v) at a flow rate of 20 mL/min. After one flash chromatography run, the purity of DISS reached over 98.0% with a recovery yield of 70.59% and a productivity of 2.35 g/h/L. The total recovery yield of DISS reached 66.59%. The preparation method is simple, efficient, energy-saving and proper for large scale preparation of DISS from Polygalae Radix. (C) 2014 Elsevier B.V. All rights reserved.