<p>Aconitum alkaloid poisoning which can lead to malignant arrhythmia, cardiogenic shock and even death has been frequently reported due to improper intake of <i>Aconitum</i> species in hospital emergency rooms. In this study, a simple method for simultaneous determination of 14 aconitum alkaloids in human blood and urine by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was developed. Blood and urine samples were purified by supported liquid extraction (SLE) and analyzed by UPLC-MS/MS. Good linear relationships for 14 <i>aconitum</i> alkaloids were obtained in the corresponding range of 0.1–50&#xa0;ng&#xa0;mL⁻¹ with correlation coefficients greater than 0.999 and detection limits of between 0.002 and 0.019&#xa0;ng&#xa0;mL⁻¹. The average recoveries measured at three concentration levels ranged from 80.0% to 110.1% and 85.0% to 110.2% in spiked blood samples and urine samples, respectively, with the relative deviation less than 10%. The blood matrix effects ranged from 88.3% to 109.8%, while the urine matrix effects ranged between 91.4% and 103.6%. Both the intra-day and inter-day precisions were less than 11%. Finally, the method was successfully applied to the determination of <i>aconitum</i> alkaloids in the blood and urine of clinic poisoned patients showing its good potential for poison diagnosis in clinical emergency cases.</p>