The invention relates to a separation and purification method for ingenol monomers, and belongs to the technical field of the separation and purification for the active ingredients of traditional Chinese medicines. The separation and purification method disclosed by the invention comprises the following steps: by taking the traditional Chinese medicine moleplant seeds of euphorbia as raw materials, performing ethanol solution extraction, ethyl acetate extraction, silica-gel column chromatography, methanol dissolution and filtration, preparative high-performance liquid chromatographic separation and product recovery to obtain ingenol monomer products. According to the separation and purification method disclosed by the invention, the content of ingenol in the products achieves more than 98% by virtue of the optimum process and parameter conditions; the whole technical process is stable, simple and convenient to operate, high in separation efficiency, and low in cost, and is capable of separating and preparing the ingenol monomers with a high purity.