The invention provides a method and a kit for simultaneously detecting five steroid hormones and application of the method and the kit, and belongs to the technical field of steroid hormone detection. The method comprises the step of detecting a pretreated sample to be detected on the basis of LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry). The pretreatment method is optimized, so that accurate detection of five steroid hormones including testosterone, androstenedione, 17alpha-hydroxyprogesterone, dihydrotestosterone and dehydroepiandrosterone sulfate is realized, and the method can be applied to detection items of important steroid hormones in clinical androstenemia. The real level of the real concentration of the five steroid hormones in the body of a subject can be given easily, clinical use or laboratory use is facilitated, and the method has important guiding significance on auxiliary diagnosis of hyperandrogenemia and polycystic ovarian syndrome and has high practical application value.