Abstract: For the simultaneous and accurate determination of important small-molecule metabolites （including amino acids, and neurotransmitters） in the human body, and to provide a technical reference for the diagnosis of disease occurrence and development, ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） was employed to determine the contents of 20 amino acids and 6 neurotransmitters in the serum from healthy individuals and type 2 diabetic patients. The t-test was used to screen for compounds with significant differences. Frozen human serum samples were thawed at 4 ℃ and diluted 20 times with water. An aliquot （10 μL） diluted serum or serum was taken, and mixed with 390 μL of acetonitrile solution containing 10 mmol·L⁻¹ hydrochloric acid. The mixture was vortexed for 5 min, and centrifuged for 10 min, and the supernatant was taken for UHPLC-MS/MS analysis. The Waters Acquity UPLC BEH Amide chromatographic column was used as the stationary phase, with the mobile phase system of 0.2% （volume fraction, same below） formic acid solution-acetonitrile solution containing 0.2% formic acid （for 18 target compounds） or 0.2% formic acid solution containing 5 mmol·L⁻¹ ammonium acetate-acetonitrile solution containing 0.2% formic acid （for 8 target compounds） for chromatographic separation. Electrospray ion source in positive and negative ion modes was used for ionization, multiple reaction monitoring mode was used for detection, and matrix-matched method was used for quantification. It was shown that linear relationships between the mass concentrations and peak areas of the 26 target compounds were kept in definite ranges, with detection limits in the range of 0.4–1 000 μg·L⁻¹. Test for recovery was performed using the standard addition method, giving recoveries in the range of 87.4%–113%, with RSDs （n=6） of the determined values ranging from 0.10% to 12%. The proposed method was applied to the analysis of serum from healthy individuals and type 2 diabetic patients, 20 target compounds were detected, and 13 compounds with significant differences were screened.