Coupled with evaporative light scattering detection, a high-speed countercurrent chromatography method was successfully applied to the separation and purification of three tauro-conjugated cholic acids from the traditional Chinese medicine Pulvis Fellis Suis. With a two-phase solvent system composed of chloroform–methanol–water–acetic acid (4:4:2:0.3, v/v), taurochenodeoxycholic acid (10.2 mg), taurohyodeoxycholic acid (11.8 mg) and taurohyocholic acid (5.3 mg), were obtained from 100 mg of the crude extract with purities of 94.6, 96.5 and 95.4%, respectively, in one-step separation. The structures of the three tauro-conjugated cholic acids were identified by ESI-MS, 1H NMR and 13C NMR.