This invention discovers a purification method for isolating an extract containing chlorosesamone from roots of genus Sesamum plants, and its application. In this invention, D2 fraction containing chlorosesamone is obtained through an extraction step and a separation step. Then, through further purification step, chlorosesamone is obtained. In the experiment of this invention, D2 fraction or chlorosesamone extracted from roots of Sesamum indicum, a plant of genus Sesamum, can be used for the anticancer treatment for cancer cells of breast, liver, blood and lung.