The interaction of Coomassie brilliant blue G-250 (CBBG-250) with bovine serum albumin (BSA) was investigated by the methods of ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), and molecular modeling technique. The UV-Vis results showed that BSA could interact with CBBG-250 to form a CBBG-BSA complex in a molar ratio of 1:1 with increasing BSA concentrations. At different temperatures, the apparent binding constant (Ka) values were 5.03 ? 104 (298 K), 3.04 ? 104 (303 K), 2.84 ? 104 (308 K), and 1.99 ? 104 L/mol (313 K), respectively, which decreased with increasing temperatures. The whole process was enthalpically and entropically driven, and the enthalpy (△H) and entropy changes (△S) were 45.32 kJ/mol and 139.18 J/(mol?K), respectively, indicating that the hydrogen bonds and Van der Waals forces played a dominant role in the molecular interaction. FT-IR was used to study the changes in characteristic amide bands (I and II) of α-helix in BSA before and after the interaction. The results showed that amide band I shifted from 1650 cm-1 to 1710 cm-1, amide band II shifted from 1573 cm-1 to 1544 cm-1, and the α-helical structure content was reduced. CD measurements and molecular docking technique further verified the changes in BSA structure and the content of α-helical structure decreased from 42.15% to 1.27%.