The invention relates to a molecular marker related to azoospermia and a detection method and application thereof, and belongs to the technical field of analytical chemistry and clinical medicine. Metabolic small molecules in normal control and azoospermia semen are compared by adopting UPLC-Qexactive MS (Ultra Performance Liquid Chromatography-Quantitative Active Mass Spectrometry), 1, 3-dihydroxypropyl-2-yl pentadecanoate which can be used for evaluating whether the semen suffers from azoospermia or not and has diagnostic value exists in the semen, and the application of the UPLC-Qexactive MS for detecting the 1, 3-dihydroxypropyl-2-yl pentadecanoate is found, so that the 1, 3-dihydroxypropyl-2-yl pentadecanoate can be used for evaluating whether the semen suffers from azoospermia. And the azoospermia diagnosis and monitoring kit convenient for clinical application is developed. Metabolic small molecules are novel biomarkers, are highly associated with disease outcomes, and are stable, non-invasive, easy to detect and accurate in quantification. The 1, 3-dihydroxypropyl-2-yl pentadecanoate provided by the invention can be used as a diagnostic marker of azoospermia, provides a basis for further deep examination of clinicians, provides support for quickly and accurately mastering the disease state and the severity of the disease state of a patient and timely taking a prevention and treatment scheme, and delays and prevents disease progression.