Belonging to the field of analytical chemistry and clinical medicine, the invention discloses an idiopathic male infertility related steroid hormone marker in serum, a detection method and application thereof. The marker is cortisol and/or dehydroepiandrosterone, a UPLC-Q exactive MS method is employed for detection, the marker can be used for auxiliary diagnosis and monitoring of idiopathic male infertility, has high sensitivity and specificity, and has clinical promotion value.