The invention discloses a method for detecting GABA, Glu, DA, 5-HT and amphetamine type stimulants from serum. The method comprises steps as follows: S1: preparation of a standard liquid: GABA, Glu, DA, 5-HT and amphetamine type stock solutions, standard curve working solutions and quality control working solutions are prepared, and an internal standard compound stock solution and a working solution are prepared; S2: treatment of blank serum samples and a drug addict serum sample: the standard curve working solutions or the quality control working solutions of GABA, Glu, DA, 5-HT and amphetamine type compounds are added to the blank serum samples respectively, then a trichloroacetic acid containing Glu-d5 and a acetonitrile solution containing DA-d4 and Amp-d6 are added sequentially, vortex shocking and centrifugation are performed, and supernatants are sucked for testing; except addition of the standard curve working solutions or quality control working solutions, an unknown biological sample is subjected to the treatment manners the same as above; S3: liquid chromatograph-mass spectrometer detection: the supernatants prepared in the S2 are detected; S4: drawing of standard curves. The method has the characteristics of convenience in operation, high efficiency and practicability.