The invention relates to the technical field of biological medicine. In particular, the invention relates to a fomes fomentarius compound extract, a preparation method and an application thereof. The fomes fomentarius compound extract is prepared from the following components according to a weight ratio of 1-10 parts of fomes fomentarius:1-9 parts of phellinus pini:1 part of ganoderma lucidum; the extraction method of the compound extract comprises: preparing a fomes fomentarius compound, mixing, crushing, adding pure water, performing water extraction and alcohol precipitation to obtain a supernatant, performing reduced pressure concentration, and performing vacuum drying of the concentrate to obtain the product. The content of triterpene and triterpene saponin in the fomes fomentarius compound extract of the invention is not less than 5.5%. The extract not only has significant in-vivo and in-vitro antineoplastic efficacy, but also has no obvious acute toxicity; the maximum tolerance dose for mice is 0.2 g (equivalent to 4.58 g of crude drugs), which is 327 times of human oral dosage. The extract can be prepared into compound preparation when mixed with pharmaceutically acceptable carriers, and the compound preparation can be oral liquid, injection, suspension, pills, tablets, capsules, powder, granules, or drop pills.