Abstract: Abstract: Lactic acid bacteria have a long history of safe use in food. Among them, the antifungal properties of Lactobacillus plantarum are particularly interesting. Numerous studies have pointed out that the natural compounds produced by L. plantarum can significantly inhibit the growth of fungi and Aspergillus flavus, spores, thereby degrading aflatoxin, especially for the longer shelf life of a variety of food. Moreover, the acid is attributed to the antifungal activity of L. plantarum, where organic acid is the most important acidic metabolite of L. plantarum. In addition, acetic acid is the most effective antifungal metabolite among the many organic acids produced by L. plantarum. At the same time, there is the highest content of lactic acid, acetic acid, and propionic acid in the L. plantarum fermentation supernatant organic acid. However, the inhibitory effect of L. plantarum on A.flavus is mostly focused on the antifungal substances produced by a single strain in recent years. Only a few studies were on the differential metabolites of multiple strains with different antifungal activities. Moreover, standard products are still lacking to verify the activity of inhibiting A.flavus on the quantitative amount of lactic acid, acetic acid, and propionic acid in the supernatant of L. plantarum. Therefore, the purpose of this research is to find the small molecular metabolites that inhibit A.flavus through the comparison between multiple strains, with emphasis on the practical application of L. plantarum. 16 strains of L. plantarum with different antifungal activities were selected, 8 strains of which presented strong antifungal activity (Strong group) and the rest was weak (Weak group). Ultra-high performance liquid chromatography-quadrupole flight Time mass spectrometry combined with PCA (Principal Components Analysis) and OPLS-DA (Orthogonal Partial Least Square-Discriminate Analysis) was utilized to explore the different metabolites between strains with different antifungal activities, further to determine the substances with main antifungal effects. At the same time, standard products of 1, 2, and 3 times the concentration were used to verify the inhibit activity of A.flavus, according to the quantitative determination for the content of lactic acid, acetic acid, and propionic acid in the fermentation supernatant of L. plantarum in the early stage of the laboratory. The results showed that there were significantly different metabolites in the two groups of L. plantarum fermentation supernatants (P<0.05). Database comparison demonstrated 30 significantly different metabolites, including imidazoleacetic acid, tyrosine were identified (P<0.05). Among them, acidic substances were relatively different, such as organic and fatty acids. Correspondingly, the acidic substance in the supernatant was attributed to the main antifungal effect, whereas the antifungal activity depended on the acidic environment of low pH value. Lactic acid, acetic acid, and propionic acid presented excellent antifungal activities among organic acids produced by L. plantarum. Specifically, the antifungal activity was ranked in order of propionic acid>acetic acid>lactic acid. Comprehensively, organic and fatty acids are widely expected to be the main antifungal substances, where the L. plantarum antifungal activity increased with the concentration of the acidic substances.