The fluorescence quenching reactions of barbaloin with bovine serum albumin (BSA) in pH 7.20 Tris–HCl buffer solution were studied. The quenching mechanism of BSA by barbaloin was interpreted using the Stern–Volmer (S–V) mechanism. The binding constant K values were 2.78×10⁵ (293 K), 1.87×10⁵ (310 K), 1.25×10⁵ (318 K), and the number of binding sites (n) were 1.18, 1.14, and 1.09, respectively. In addition, the thermodynamic functions enthalpy (ΔH°) and entropy (ΔS°) for the reaction were also calculated according to Vant's Hoff equation were −23.7 kJ/mol and 23.6 J/mol, respectively. Plausible explanations of the quenching mechanism are discussed on the basis of a hydrophobic interaction between barbaloin and BSA.