Longjing tea is renowned for its fresh aroma and high value. However, during storage, the emergence of an off-flavor known as "stale odor" can significantly impact the flavor quality and economic benefits of Longjing tea. Yet, the specific volatiles responsible for this stale odor in Longjing tea remain unknown. In this study, Longjing tea samples with varying degrees of stale odor intensity were analyzed using simultaneous distillation extraction coupled with gas chromatography-mass spectrometry (SDE-GC–MS). Through odor activity value (OAV) and fractional omission testing, hexanoic acid and trans-2-nonenal were identified as the primary contributors to the stale odor. Moreover, the concentration of hexanoic acid was found to be valuable in predicting the intensity of the stale odor in Longjing tea. The oxidative degradation of linoleic acid was proved as the generation pathway of stale odor in Longjing tea. These findings provide essential theoretical principles for Longjing tea production and preservation.