The invention relates to a preparation method and application of an anticancer compound. The preparation method comprises the following steps of: heating dry serrate rabdosia herb leaves and ethanol in an amount which is 2 to 4 times weight of the dry serrate rabdosia herb leaves until boiling, performing refluxing extraction, filtering to obtain supernate, concentrating under reduced pressure to make the concentrated supernate 50 to 70 percent of the original supernate to obtain a concentrated solution, and freeze-drying to obtain powder; dissolving the power in water, loading on a D101 macroporous resin column, eluting by using an ethanol solution, collecting eluant, freeze-drying to obtain powder A; dissolving the powder A in water, loading on an HP-20 macroporous resin column, eluting by using a 70 to 90 percent ethanol solution, collecting eluant, and freeze-drying to obtain powder B; and dissolving the sample B in methanol to ensure that the concentration is 5-50mg/ml, performing gradient elution by using a reversed phase C18 column, collecting a compound peak eluted by 40 percent of water and 60 percent of methanol. The compound is applied to preparation of antitumor medicines, and has toxicity for human liver cancer cell lines, human leukemia cell lines and human breast cancer cell lines.