The interaction of coomassie brilliant blue G-250 (CBBG-250) with bovine serum albumin (BSA) was investigated by the methods of fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). Fluorescence data showed that the quenching of BSA by CBBG-250 was result of forming the complex of BSA-CBBG-250, and CBBG-250 effectively quenched the intrinsic fluorescence of BSA via static quenching. According to Stern-Volmer equation, the binding parameters between CBBG-250 and BSA were determined. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -4.38 kJ·mol-1 and -6.16 J·mol-1·K-1, indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding. The conformational investigation revealed the α-helical structure was decreased and the polypeptides of BSA were slightly folded with the addition of CBBG-250 by synchronous fluorescence. Fourier infrared spectrum showed that the peak position of amide band Ⅰ (1 600～1 700 cm-1) and amide band Ⅱ (1 600～1 500 cm-1) of the BSA characteristic absorbing peaks was blueshifted. The amide band I moved from 1 650 to 1 710 cm-1, and amide band Ⅱ moved from 1 573 to 1 544 cm-1, which indicated the structure of alpha helix features (1 650～1 658 cm-1) and beta-fold (1 620～1 640 cm-1 and 1 645 cm-1) had changed. The content of α-helical structure decreased from 42.15% to 1.27% by circular dichroism. The main reasons were the microenvironment polarity of tryptophan changed with the addition of CBBG-250 increased, two interaction between BSA and CBBG-250 including the hydrogen bonding and van der Waals force occurred, and then the secondary structure of BSA was changed, which was confirmed by Molecular modeling.