Abstract: To develop a non-targeted analytical method based on liquid chromatography-high resolution mass spectrometry (LC‒HRMS) for natural flavors, the effects of sample pretreatment method and chromatography-MS conditions on the detection were systematically investigated, and the detection results of the non-targeted analytical method was further evaluated. The results showed that: 1) A ternary solvent combination of Vmethanol׃Vacetonitrile׃Vwater=35׃35׃30 was used for ultrasound for 20 min to achieve better extraction results, and the operation was convenient. 2) The single-stage gradient elution was performed on the complementary C18 and Amide columns, in which 0.1% (volume fraction) formic acid was added to the mobile phase of the C18 column, and 0.1% formic acid and 5 mmol/L ammonium acetate were added to the mobile phase of the Amide column; while the effects of injection volume and column temperature were not obvious, which were set at 2 µL and 30 ℃, respectively. 3) The preferred MS conditions were separate scans for positive and negative ion modes, Full MS scans in the range of m/z 70~1 050 with a resolution of 60 000, and the normalized collision energies of 20%, 40% and 60%. 4) All the total ion chromatograms obtained from the multi-needle injection under the “dual-column+dual-mode” detection conditions showed good overlap, and the complementary combination of the reversed-phase and hydrophilic modes of the two columns as well as the synergistic positive and negative ion detection modes could effectively improve the coverage of compound identification. The developed non-targeted analysis method based on the “dual-column+dual-mode” for natural flavors showed high detection throughput and good stability, and is suitable for quality control and compositional analysis of complex natural flavors.