Objective The high resolution mass spectra database of common alkaloids were established and applied to the automatic screening of poisoning samples. Methods The accurate molecular mass and appropriate collision energy of each alkaloid was obtained by injecting alkaloid standard solutions into the Q exactive mass spectrometry directly or separated by high performance liquid chromatography and mass spectrometry analysis. The chromatographic analysis was performed on a HSS T3 column(2.1 mm×100 mm,1.8 μm)by gradient elution using methanol and 5 mmol/L ammonium acetate containing 0.1% formic acid solution as mobile phase. The secondary mass information of alkaloid was collected in positive electrospray ionization and targeted-selected ion monitor-ddMS² mode. The mass spectrometry data were applied to the automatic screening of actual samples. Results The mass spectrometry database of 72 alkaloids were established containing the theoretical accurate molecular mass,retention time collision energy and fragment ions. The multiple alkaloids in actual samples were locked rapidly by automatic screening with the mass spectra database. Conclusion This method is simple, fast, accurate and suitable for the rapid automatic screening of alkaloids in poisoned food sample.