The invention discloses a wedelia trilobata anti-tumor extract and preparation and application methods thereof. The wedelia trilobata anti-tumor extract is prepared by adding solvent into wedelia trilobata powder for extraction, filtering and decompression-concentrating extracted liquor to obtain a total extract, mixing the total extract with water into a suspension, performing extraction sequentially through petroleum ether and ethyl acetate and performing decompressed recovery to obtain a petroleum ether solution, an ethyl acetate solution and a aqueous solution, wherein the petroleum ether solution contains one eudesmene type sesquiterpene lactone, five enantiotopic kaurane type diterpenoid compounds, two oleanane type triterpenes, one cycloartane type triterpene, one sterol compound, two dipeptide compounds and one caffeate compound. The prepared wedelia trilobata anti-tumor extract is high in yield, simple in technical process, low in cost and applicable to large-scale preparation, and meanwhile, has broad-spectrum anti-tumor activity and can serve as an active component applied to tumor control and treatment drugs.