Abstract In order to examine the mechanism and process of sonodynamic reaction, the chlorophyll-magnesium-metronidazole (Chl-Mg-MTZ) was synthesized and characterized. Chl-Mg-MTZ acting as a new sonosensitizer was irradiated by ultrasound, and the generation of reactive oxygen species (ROS) was detected by the method of Oxidation-Extraction Spectrometry (OES). That is, under ultrasonic irradiation in the presence of Chl-Mg-MTZ, the 1,5-diphenyl carbonzide (DPCI) is oxidized by generated ROS into 1,5-diphenyl carbonzone (DPCO), which can be extracted by mixed organic solvent and display a obvious visible absorption at 563nm wavelength. Besides, the generation conditions of ROS were also reviewed. The results demonstrated that the quantities of generated ROS increase with the increase of ultrasonic irradiation time, Chl-Mg-MTZ concentration and DPCI concentration. Finally, several radical scavengers (l-Histidine (His), 2, 6-Di-tert-butyl-methylphenol (BHT) and Vitamin C (VC)) were used to determine the kind of the generated ROS. It was found that at least the hydroxyl radical (·OH) and singlet oxygen (1O2) were generated in the presence of Chl-Mg-MTZ under ultrsonic irradiation. It is wish that this paper might offer some valuable references for the study on the mechanism of SDT and the application of Chl-Mg-MTZ in tumor treatment.