The cleavage pathways of eugenol were investigated by negative electrospray ionization mass spectrometry (ESI-MS/MS). The relative molecular mass of ［M-H］- was obtained by ESI-MS/MS, and the cleavage pathways information of ［M-H］- ion were obtained from collision-induced dissociation (CID). It was found that precursor ion ［M-H］- mainly fractured through methoxy group at the C2 of benzene ring, and then continuously lost vinyl at the C4 of benzene ring with generated characteristic fragment ion at m/z 121. The fragment ion at m/z 121 may lose CO with generated characteristic fragment ion m/z 93. These three ions are the characteristic ions of eugenol, which may be used to identify eugenol. These cleavage rules can provide reference information for identification for metabolites of eugenol in aquatic products.