Hollow fiber liquid–liquid phase microextraction followed by high-performance liquid chromatography was employed to research the binding of four furocoumarin compounds (psoralen, oxypeucedanin, imperatorin and isoimperatorin) to bovine serum albumin. The results reveal that the percent of drug–protein binding did not rely on the furocoumarins and protein concentrations, and four furocoumarins scarcely competed for protein binding sites. The protein-binding percents of psoralen, oxypeucedanin, imperatorin and isoimperatorin were 55.1 ± 4.5, 14.4 ± 2.4, 44.1 ± 6.7, and 41.3 ± 3.8%, respectively, the numbers of binding sites were 1.2, 1.6, 0.6 and 0.3, and the binding constants were 1,801, 212, 937 and 4,025 L mol−1, respectively. The proposed new approach is simple, rapid, and effective for the study of the interaction between protein and furocoumarin compounds which is either individual or coexistent with the similar ones.