The interaction between Dy(Ш)/Rutin complexes and bovine serum albumin (BSA) was studied by fluorescence, ultraviolet (UV) absorption, three-dimensional fluorescence and circular dichroism spectroscopies under simulative animal physiological conditions. It was proved that the quenching process of Rutin–Dy by BSA was static quenching which promoted the formation of Rutin–Dy–BSA complexes. The binding constant KA and corresponding thermodynamic parameters at different temperatures were calculated. The results indicate that electrostatic effects are the predominant intermolecular forces in stabilizing the complex. According to Förster's theory of non-radiative energy transfer, the distance r = 2.18 nm between the donor (BSA) and the receptor (Rutin–Dy) was obtained. The energy transfer between BSA and Rutin–Dy is intramolecular. The synchronous fluorescence, ultraviolet (UV)-absorption, three-dimensional fluorescence and circular dichroism studies revealed that the conformation of BSA changed in the binding reaction, leading to some micro-environmental and conformational changes of BSA molecules and reducing of the helical structure of BSA. This study will provide valuable insights to broaden the application of Rutin in the field of medicine. [ABSTRACT FROM AUTHOR]