Rosavin was a major active compound from Rhodiola rosea. It is known that it possesses stimulant, antidepressant, and anticancer properties. In the present study, a two-step flash column chromatography has been developed for the large-scale preparative separation and purification of rosavin from the extracts of Rhodiola rosea, which were obtained by microwave assisted extraction (MAE) using ion liquids (ILs) as extraction solvent under the optimized conditions (microwave power 1200 W, extraction time 6 min, solid-liquid ratio 1:20, microwave temperature 120 degrees C) resulting from the analysis of the D-optimal design. In the first separation, the Rhodiola rosea extract was loaded into polyamide and HPD-200 macroporous resin flash columns in series for efficient enrichment of rosavin. In the second one, the rosavin-rich crude sample after the serial column chromatography was subjected to final purification by silica gel flash chromatography. After the two-step separation, 529.1 mg of rosavin was obtained at a purity of 98.2% and a recovery of 60.6%. The separation process can provide a new method for large-scale separation and purification of rosavin for its pharmaceutical and practical use.