Objective To establish a method for the isolation and preparation of isoquercetin by high speed counter-current chromatography (HSCCC). Methods The total flavonoids in Gossypii Flos extract was further separated by HSCCC with a two-phase solvent system composed of ethylacetate-water (1:1) stationary phase. Main engine speed was 850 r/min and the detection wavelength was 254 nm at a flow rate of 2.0 mL/min. The obtained fraction was identified by spectral analysis and the purity was analyzed by TLC and HPLC. Results Isoquercetin reference substance with the purity of 99% under this given situation was obtained. Conclusion The results indicate that HSCCC is a simple and rapid method for the separation and preparation of isoquercetin reference substance.