The previous research results of our laboratory showed that clove had the strongest antioxidant activity in the state’s list of food-medicine herbs promulgated. It has been found that the antioxidant activity of natural products is closely related to antiglycation activity. Therefore, this study aimed to investigate further the antiglycation activity of clove essential oil (CEO) and the interaction between eugenol the component with the highest content in CEO and bovine serum albumin (BSA). The spectral results showed that in the non-enzymatic glycation incubation system of low-density lipoprotein (LDL), CEO had significant inhibition effects on forming the early, intermediate and late products of LDL glycation and had the strongest inhibition effect on the late product. The composition analysis of CEO by gas chromatography-mass spectrometry (GC-MS) indicated that eugenol was the most abundant component in CEO. Multispectral and molecular docking were applied to investigate the interaction between eugenol and BSA. The result of ultraviolet-visible (UV-Vis) absorption spectroscopy indicated an interaction between eugenol and BSA. In fluorescence emission spectroscopy, with the increase of eugenol concentration, the fluorescence intensity of BSA gradually increased with the blue shift, which further proved the interaction between them. The calculated results of binding parameters at different temperatures confirmed that the thermodynamic processes were involved in the interaction between eugenol and BSA. Thermodynamic parameters and the site marker competitive experiments showed that eugenol particularly bonds to BSA at the site Ⅰ through hydrogen bond and vander Waals force. In synchronous fluorescence (SF), three-dimensional (3D) fluorescence, and Fourier transform infrared (FTIR) spectroscopy, the signal strengths changed with the increase of eugenol concentration, and the shifts also occurred, which indicated that the conformation of BSA changed with the addition of eugenol. The results of the interaction between eugenol and BSA were further verified by molecular docking technology. The findings can provide theoretical support for the further development of clove.