The invention belongs to the field of analytical chemistry and clinical medicine and discloses idiopathic male infertility diagnostic markers serine and sorbitol in seminal plasma as well as a detection method and application thereof. The markers are serine and/or sorbitol and are detected by adopting a UPLC (Ultrahigh Performance Liquid Chromatography)-Qexactive MS (Mass Spectrum) method; and the markers can be used for auxiliary diagnosis and monitoring of idiopathic male infertility, have relatively high sensitivity and specificity and have a clinical popularization value.