The invention discloses a double tetrahydrofuran lignin, a preparation method and an application thereof. The preparation of the lignin comprises the following steps: (1) crushing clinacanthus nutans and then soaking in ethyl alcohol, refluxing and extracting, thereby acquiring total ethyl alcohol extract, and then uniformly dispersing in distilled water and using petroleum ether and ethyl acetate for extracting in turn; (2) treating the petroleum ether part extract with silica gelcolumn chromatography, adopting a petroleum ether-ethyl acetate solvent system for gradient eluting, collecting, concentrating and mixing the same flow components; (3) performing reverse phase silica gel column chromatography separation, Sephadex LH-20 sephadex column chromatography separation and high performance liquid chromatography separation on the flow component acquired by eluting with petroleum ether and ethyl acetate at ratio of 5:1, thereby acquiring the double tetrahydrofuran lignin. According to the invention, the double tetrahydrofuran lignin extracted from clinacanthus nutans has inhibitory activity to cervical cancer Hela cells, breast cancer MCF-7 cells and lung adenocarcinoma A549 cells.