Watermelon seed kernels (WSK) are prone to oxidative rancidity, while their evaluation biomarkers and changes in volatile flavor are still unknown. The research tracked the changes in volatile compounds and lipid components before and after rancidity using HS-SPME-GC-O-MS and lipidomic techniques. The results showed the flavor of watermelon seed kernels changed significantly before and after rancidity, from mild aroma to rancidity. A total of 42 volatile compounds were detected via GC-O-MS, and a total of 220 lipid molecules were detected via lipidomic technology. 55 lipids with significant differences were screened via multivariate statistical analysis. Combining the above analysis, it found that glycerol phospholipid and glyceride pathways were the most important metabolic pathways and 1-Pentanol and styrene could be used as potential biomarkers to judge the rancidity process of watermelon seed kernels. The research could provide powerful technical support for the storage, transportation and freshness preservation of watermelon seed kernels.© 2024 The Authors.