Abstract: In order to investigate the changes of volatile flavor substances in the process of beeves spoilage, beeves with different storage time were employed. The volatile flavor substances of beeves in spoilage process were analyzed by using gas chromatography ion transfer spectroscopy (GC-IMS) and electronic nose. The results showed that a total of 55 volatile flavor substances were detected through GC-IMS, mainly including 12 ketones, 10 alcohols, 9 esters, 8 aldehydes and 4 hydrocarbons. GC-IMS and electronic nose analysis indicated that the types and contents of volatile flavor substances significantly increased during the spoilage process. The alcohols, aldehydes, esters, acids, furans, pyrroles, thiophenes, amines, pyridines and ethers continuously increased during the spoilage process. However, ketones, hydrocarbons, and sulfur compounds gradually increased and reached peak at the seventh day, and then gradually decreased. PCA and PLS-DA analysis suggested that there were significant differences in volatile flavor substances of beeves in spoilage process. Sixteen different volatile flavor substances were identified by variable importance for the projection (VIP) value, with the extension of storage, the contents of irritating and unpleasant flavor substances such as n-hexanal-D, n-hexanal-M, butyraldehyde-M, butyraldehyde-D, tetrahydropyrrole-M, isoamyl-D, isoamyl-M, n-propanol-D, 2,3-butanedione gradually increased, which could be used as potential biomarkers to distinguish beeves with different degrees of spoilage. This work provides a theoretical basis for monitoring beef spoilage process.