Flavonol isomers, which differ only in the position of hydroxyl group substitute on their chemical structures, showed different chromatographic behaviors. These polar compounds, in general, can be analyzed by high performance liquid chromatography. In this paper, the separation of flavonol isomers by home-set up packed column supercritical fluid chromatograph with carbon dioxide modified by ethanol containing 0.5% (V/V) phosphoric acid as mobile phase is described. The effects of temperature, pressure, composition of mobile phase and packed column type on the separation were studid. The addition of phosphoric acid to the mobile phase, which can not result in corrosion on the chromatographic system, enabled flavonol to elute from the column, and showed that the composition of the mobile phase was the most pronounced factor influencing the separation. Three columns were evaluated for the separation. It showed that the phenyl-bonded silica column was the best and the ODS column was the worst for the isomers separation. Increasing pressure shortened the retention time of each compound with good resolution, and higher temperature led to longer retention time.