The invention provides an inula wissmannian extract and preparation thereof and application in preparation of an antitumor drug. The extract is gemma alkane type sesquiterpene lactone inula wissmannian lactone methyl, ethyl, propyl, butyl and amyl or sheepear inula herb lactone. The extract has the following chemical structural formula and absolute configuration that: due to the toxicity killing function experiment of inula wissmannian lactone methyl, ethyl, propyl, butyl and amyl or sheepear inula herb lactone on human hepatoma cells HepG2, human prostatic cancer cells PC-3, human gastric carcinoma cells MGC-803, human leukemia cells K562, human nasopharynx cancer cells KB and normal human hepatocytes LO2, the result shows that the gemma alkane type sesquiterpene lactone has strong tumor cell killing function, and proliferation of tumor cells can be inhibited, so that the tumor cells are killed. The animal in-vivo experiment proves that the inula wissmannian plant extract and monomeric compounds have the anti-tumor effect and can serve as active ingredients for preparing the antitumor drug. A novel cancer treatment medicine is provided clinically, and high clinical application values and social benefits are generated.