The present invention pertains to the field of chemical analysis and detection, and particularly relates to a pretreatment method for LC-MS detection of Aspergillus flavus metabolomics. The method comprises: culture of Aspergillus flavus strains; quenching of Aspergillus flavus; disruption of the cell envelope and extraction of the metabolome of Aspergillus flavus; and analysis of the metabolome of Aspergillus flavus. A cold glycerol buffer solution is used in combination with a rapid suction filtration method for quenching, and a MeOH/DCM/ACN/EA/HCOOH mixed solution is used as an extraction solution, achieving the objective of efficient extraction of different polar compounds, and producing high metabolome compound coverage. Pretreatment of Aspergillus flavus cell metabolomics using the method can ensure the repeatability and stability of a metabolomics analysis method and reduce false positive rates of test results.