Theaflavin is a high-quality component of black tea， which has good medical and health functions. High-speed countercurrent chromatography (HSCCC) has been applied for the separation of theaflavins and catechins， and examined the cytotoxic activity of the individual theaflavins against human liver cancer (HepG2) cells， Colonic adenocarcinoma(Caco-2)cells. Six components were separated and prepared by HSCCC. Further analysis of these compounds by HPLC indicated that that each component F4 contains theaflavin(TF)， F5 contains theaflavin-3-gallate(TF-3-G) and theaflavin-3'-gallate (TF-3'-G)， and F6 contains theaflavin-3，3'- gallate (TFDG). The results show TFDG was selected to study the inhibitory effect on cancer cells. CCK-8 was used to assess cell proliferation. TFDG has a significant inhibitory effect on HepG-2 and Caco-2 cells in a time-dependent and dose-dependent manner with the increase of concentration and time. It found a solvent system for the preparation of theaflavins in large quantities was selected based on the partitioning coefficient K and separation factor α. Also， we acknowledged that there is a certain relationship between cancer prevention and quinone reductase(QR). Combining experimental data with theory， by linking molecular docking technology was used to verify that TFDG and QR can form stable complexes， It also inducing QR activity can enhance the anticancer effect， it can providing a basis for further research on the effect of theaflavin on cancer cells.