This study investigated the interaction between eupatorin and bovine serum albumin (BSA) using ultraviolet-visible (UV-vis) absorption, fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopies, and molecular modeling at pH 7.4. Results of UV-vis and fluorescence spectroscopies illustrated that BSA fluorescence was quenched by eupatorin via a static quenching mechanism. Thermodynamic parameters revealed that hydrophobic and electrostatic interactions played major roles in the interaction. Moreover, the efficiency of energy transfer, and the distance between BSA and acceptor eupatorin, were calculated. The effects of eupatorin on the BSA conformation were analyzed using UV-vis, CD, and synchronous fluorescence. Finally, the binding of eupatorin to BSA was modeled using the molecular docking method.