The interaction between Gliotoxin and bovine serum albumin (BSA) was studied by the fluorescence, Circular Dichroism (CD) and ultraviolet visible (UV-Vis) techniques. The fluorescent experiment showed that the intrinsic fluorescence of BSA was quenched by the binding of gliotoxin in a static quenching procedure, with an association constant of 7.2′103 L/mol and in hydropobic forces. And the CD spectrum revealed that gliotoxin effected the conformation of BSA by increased the mass of α-helix.