[Objectives]  To establish an effective method for the separation and preparation of diene monoepoxides regioisomers mixtures of the Lepidopteran Type Ⅱ sex pheromone. [Methods]  The parameters of the method were established using forward system, semi-preparative, high performance, liquid chromatography combined with an evaporative light scattering detector and SunFire Silica OBD preparative column, after which the prepared monomers were verified by gas chromatography-mass spectrometry and field trapping. [Results]  The semi-preparative high performance liquid chromatography was eluted with 0.5 % tetrahydrofuran in hexane at a flow rate of 7.0 mL/min with the drift tube temperature of the evaporative light scattering detector set at 40 ℃ and the gain value set at 500. This method efficiently separated diene monoepoxides regioisomers mixtures of the Type Ⅱ sex pheromone with a resolution of 2.72-3.10. The purity of the prepared compounds was significant and the recovery rate of prepared samples was 80.73%-98.47%. Field trapping tests confirmed that the prepared Z3, epo6 and Z9-18:H, in combination with Z3, Z6 and Z9-18:H, specifically attracted Ectropis grisescens (45.33 moths/trap over 28 d), whereas the prepared Z3, Z6 and epo9-18:H, in combination with Z3, Z6 and Z9-18:H, specifically attracted Miltochrista striata (44.00 moths/trap over 28 d). [Conclusion]  Semi-preparative high performance liquid chromatography is a simple, convenient and efficient method for the separation and preparation of diene monoepoxides regioisomers mixtures of the Lepidopteran TypeⅡ sex pheromone, and prepared samples can be used for sex pheromone studies and small-scale field applications.