The invention discloses a preparation method of a drypetes congestiflora stem extract. The preparation method comprises the steps: adding a solvent in dried drypetes congestiflora stem powder for extracting; and performing filtration and vacuum concentration on an extraction solution to obtain a total extract, adding water for suspending, sequentially extracting by using petroleum ether, chloroform, ethyl acetate and normal butanol, and performing decompression recovering to respectively obtain a petroleum ether part, a chloroform part, an ethyl acetate part and a normal butanol part, wherein the petroleum ether part comprises three triterpene compounds, 7 sterols compounds and one glyceride derivative. The extract prepared by adopting the preparation method is high in yield, simple in process, low in cost, suitable for larger-scale preparation, and good in anti-tumor activity, and can be applied to medicines for preventing and treating tumors.