The invention provides a novel culture medium for producing novel antitumor active compound chetracin B by virtue of fungus Oidiodendron truncatum, wherein each liter of the novel culture medium consists of 2-10g of maltose, 0.5-5g of peptone, 1-10g of corn flour, 0.5-10g of sodium glutamate, 10-100g of glucose, 0.2-4g of a yeast extract, 0.1-1g of magnesium sulfate heptahydrate, 0.1-3g of potassium dihydrogen phosphate and the balance of de-ionized water. The invention also provides a preparation method of the culture medium. The culture medium disclosed by the invention is easy in preparation of raw materials, relatively low in cost, simple in preparation method, and capable of significantly promoting the yield of chetracin B produced from the fermentation of Antarctic-derived fungus GW3-13, so that the chetracin B yield is increased to 54.7mg/L from original 5.6mg/L; the culture medium is conducive to large-scale industrial production, and is of great significance for studies on the fermentation of the fungus GW3-13 and for the medicine researches of the chetracin B.