The invention belongs to the field of analytical chemistry and particularly relates to a detecting method for determining various neurotransmitters on the basis of in situ derivation. The method is characterized in that 4'-carbonyl chloride-rhodamine is used as the derivative reagent for the first time, the ultrasonic assistance-in situ derivation-dispersive liquid-liquid microextraction technology is used to derive amino acid and monoamine neurotransmitters, and the ultra-high performance liquid chromatography triple quadrupoles tandem mass spectrum with multiple reaction monitoring modes is used to perform qualitative and quantitative detection; three internal standard substances (isoprenaline, 5-ketoindole-2-carboxylic acid and norleucine) are used for building the linear equations of the neurotransmitters of three chemical structures; the existence of the neurotransmitters is accurately determined by quantification through an internal standard method. The detecting method is simple, fast, high in sensitivity, good in selectivity, good in recovery rate, capable of fast and accurately determining the neurotransmitters, and the like.