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Abstract: In this paper, the bovine serum albumin (BSA) was selected as a target molecule, the sonodynamic damage to protein in the presence of promethazine hydrochloride (PMT) and its mechanism were studied by the means of absorption, fluorescence and circular dichroism (CD) spectra. The results of hyperchromic effect of absorption spectra and quenching of intrinsic fluorescence spectra indicate that the ultrasound-induced BSA molecules damage is enhanced by PMT. The damage degree of BSA molecules increases with the increase of ultrasonic irradiation time and PMT concentration. The results of synchronous fluorescence, three-dimensional fluorescence and CD spectra confirmed that the synergistic effects of ultrasound and PMT induced the damage of BSA molecules. The results of oxidation–extraction photometry with several reactive oxygen species (ROS) scavengers indicate that the damage of BSA molecules could be mainly due to the generation of ROS and both 1O2 and e important mediators of the ultrasound-induced BSA molecules damage in the presence of PMT. [Copyright &y& Elsevier]