Apart from being used as a pharmaceutical, the inflorescences, leaves and roots of Taraxacum mongolicum are processed into different food products. However, only few phytochemical investigations on this plant have been performed. In the present study, a preparative high-speed counter-current chromatography (HSCCC) for the separation and purification of bioactive compounds from T. mongolicum was developed. Two lignans, mongolicumin A and rufescidride, were obtained. The target compounds were finally isolated and purified with a solvent system composed of ethyl acetate–n-butanol–water (2:5:7, v/v/v). The lower phase was used as the mobile phase in the head to tail elution mode. By injecting 500 mg of the enriched extract of T. mongolicum, one-step HSCCC procedure yielded 36.7 mg of mongolicumin A and 43.9 mg of rufescidride with the purity of 98.7% and 98.5%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of the two lignans were confirmed by UV, IR, HRESIMS, 1D and 2D NMR. Among them, mongolicumin A is a new compound, and rufescidride was obtained from genus Taraxacum for the first time. Furthermore, lignans were first isolated and identified from genus Taraxacum.