The invention relates to a preparation method of a Euphorbia factor L9. The Euphorbia factor L9 is extracted from Euphorbia lathyris seeds by a two-dimensional liquid chromatography technology, and complementary separation is achieved through reasonable selection of a chromatographic column, a dissolving solution, a mobile phase and the like, optimization of the flow rate, elution mode, collecting sections and other process conditions, and orthogonality of a first dimension and a second dimension, so that the whole separation and purification method is feasible, the efficiency is high, the production cycle is short, the process is stable, the operability is high, and the obtained Euphorbia factor L9 purity can be up to over 97.5%.