This experiment aimed to investigate the components of volatile substances in avermectin fermentation broth and the volatile differential metabolites across different fermentation periods. Utilizing headspace solid-phase microextraction(HS-SPME) and gas chromatography-mass spectrometry(GC/MS) non-targeted metabolomics technology in conjunction with odor activity value(OAV), the volatile components in 36 h and 168 h fermentation broths were qualitatively and quantitatively analyzed. The differential volatile metabolites were analyzed by principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA) and cluster analysis(HCA). The findings revealed that 396 volatile organic compounds were detected in the fermentation broth of avermectin, primarily consisted of hydrocarbons, halogenated hydrocarbons, alcohols, ketones, esters, heterocyclic compounds and terpenoids; among them, there were 11 substances with OAV>200, which were the key components contributing to the odor of avermectin fermentation broth. There were two distinct metabolites identified from the two fermentation periods, namely 5-methyl-2-heptanone and o-cymene.