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 V_methanol∶V_acetonitrile∶V_water=35∶35∶30 combined with 20 min ultrasound treatment could achieve better extraction results, and the operation was convenient. 2) The single-stage gradient elution was performed on the complementary C₁₈ and Amide chromatographic 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. The effects of injection volume and column temperature were not obvious, which were set at 2 µL and 30 ℃, respectively. 3) The optimized 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%, respectively. 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 interaction 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.