The invention discloses a method and a kit for detecting various related substance of CAH at the same time. The method is used for detecting 17 alpha progesterone, androstenedione, 11-deoxycortisol, 21-deoxycortisol and cortisol at the same time. The method includes: (1), adding an organic solvent into a mixed internal standard product to prepare a stock solution, and diluting to obtain extractionworking liquid; (2), using the extraction working liquid to incubate a to-be-detected blood spot sample; (3), centrifuging, taking supernate, concentrating, and using 45% methanol for re-dissolving;(4), using LC-MS/MS to detect a re-dissolving product, wherein the mixed internal standard product includes isotope internal standard products of 17 alpha progesterone, androstenedione, 11-deoxycortisol, 21-deoxycortisol and cortisol. The method can quantify five hormones in one time, can provide specific, sensitive, low-false positive and high-positive-prediction-value CAH screening and can screen CAH of 11-OHD and 21-OHD at the same time, thereby improving screening efficiency and quality.