An anti-cancer active substance from Antrodia Cinnamomea, produced from mycelium of Antrodia Cinnamomea with following steps: (1) culturing mycelium of Antrodia Cinnamomea by liquid fermentation ； (2) extracting the thus-obtained mycelium with ethanol and concentrating the extracted solution to obtain an ethanol extract ； (3) dissolving said ethanol extract in water, and extracting with equal volume of ethyl acetate to obtain ethyl acetate extrct ； (4) purifying said ethyl acetate extract by silica gel column chromatography using hexane/ethyl acetate gradient as the mobile phase to obtain an eluate fraction g ； and (5) purifying said eluate fraction g by silica gel column chromatography using hexane/ethyl acetate gradient as the mobile phase to obtain a specific eluate fraction which could be purified further to obtain said anti-cancer active substance from Antrodia Cinnamomea.
