The invention discloses a tandem mass spectrometry kit for simultaneously detecting PKU, CAH and G-6PD deficiency, comprising: a calibrator, an isotope internal standard, an incubation solution, a diluent, an enzyme quencher, a reconstitution solvent and a mobile phase; Calibrator contains phenylalanine, 17‑α‑hydroxyprogesterone, and glucose‑6‑phosphate dehydrogenase; isotopic internal standard contains phenylalanine‑d8 and 17‑α‑hydroxyprogesterone‑d8; Consists of water, buffer salts, glucose‑6‑phosphate, nicotinamide adenine dinucleotide phosphate, and surfactant; diluent consists of water, buffer salts, and surfactant; mobile phase consists of mobile phase A and mobile phase B ; Mobile phase A is an aqueous solution of ammonium formate and / or ammonium acetate; Mobile phase B is methanol and / or acetonitrile. The invention also discloses a method for simultaneously detecting PKU, CAH and G-6PD deficiency using the tandem mass spectrometry kit. The kit of the invention can detect multiple substances at the same time, and has the characteristics of high sensitivity and strong specificity.