The invention relates to a preparation method of dehydroisoandrosterone. According to the preparation method, a compound 1 is prepared by a compound 2 through esterification reaction, ketalation reaction, reduction reaction and hydrolysis reaction. When the dehydroisoandrosterone is prepared by the chemical synthetic method, the defects of difficulty in control, cumbersome post treatment, high risk, pollution of the environment, and the like in traditional production method are avoided. Compared with the traditional production method, the reaction conditions of the preparation method are easyto control, the post treatment process is simple, and the yield is higher can be up to 80.79% after refining. Raw materials of the preparation method can be prepared by sterol through microbial fermentation, the raw materials are wide in sources, are cheap and relatively environmentally friendly and can be purchased from Hunan Nuokai Biological Pharmaceutical Co., Ltd, and the structural formulasof the compound 1 and the compound 2 are as follows.