A method of non-targeted metabolomics coupled to ultra-high performance liquid chromatography-high resolution mass spectrometry was used to investigate effect of temperature on the metabolism of aspergillus flavus. Multivariate statistical analysis on the metabolomics data was carried out by using chemometric methods of OrthoPLS-DA(alternative partial least squares discriminant analysis) etc. The metabolism characteristics of aspergillus flavus were identified by MS/MS and library searching. In the study,the non-targeted metabolomics analytical method was validated by using internal standard and pooled quality control samples. The method was applied to investigate the effect of temperature on the metabolome of aspergillus flavus,and 3593(T-test,p<0.01) differential metabolites were found under different temperature conditions. Twenty candidate biomarkers were screened out. The results showed that temperature significantly affected the biosynthetic pathways of tricarboxylic acid cycle,fatty acid,phenylalanine,tryptophan and tyrosine, and regulated the secondary metabolites biosynthetic pathway enzymesactivity of aflatoxin,cyclopiazonic acid and kojic acid. This study provided a new insight for risk assessment and early warning research of aflatoxin in China.