Structure changes of bovine serum albumin (BSA) under ultrasound treatment were studied using Fourier transform infrared spectroscopy (FTIR) and fluorescence spectroscopy. The largest emission peak of BSA solution’s fluorescence spectra shifted in blue orientation, indicating that the environment of the Trp residues in BSA had altered with ultrasound treatment. The fluorescence intensity of the solution has also decreased with ultrasound, which showed fluorescence quenching effect and the conformation changes of the BSA. The relative contents of α-helix, β-fold, β-turn and random coil under different ultrasound treatment power and time were quantitatively determined via analysis of the amide Ⅰ changes of infrared spectra of BSA using curve fitting method, the secondary structure of BSA had variation trend from α-helix to β-sheet, however, the relative contents random coil had not significant change.