Abstract: In order to further confirm the sonosensitizing property of 4,4-dimethylcurcumin (DMCU), by taking the 4',6-diamidino-2-phenylindole (DAPI) as a fluorescent probe, the fluorescence and UV–Vis spectrum method is used to study the damage effect of ultrasonic irradiation combining DMCU on DNA. Meanwhile, the production and types of reactive oxygen species (ROS) during ultrasonic action are detected and identified by oxidation-extraction spectrophotometry and reactive oxygen scavengers. In addition, the effects of ultrasonic irradiation time and DMCU concentrations on the sonodynamic damage of DNA are also considered. The results show that the ultrasound combined with DMCU could damage DNA obviously, and the damage degree of DNA increases with the increase of ultrasonic irradiation time and DMCU concentrations. Additionally, DMCU is degraded under ultrasonic irradiation. The rate and extent of DMCU degradation is related to that of DNA damage under certain conditions, which indicates that the generation of reactive oxygen species in the sonochemical reaction of DMCU is responsible for the damage of DNA. The results of this study can provide a strong evidence for the inference of sonosensitizing effectiveness of DMCU.