The ozonolysis of aflatoxin B(1) (400 μg/mL) in acetonitrile solution was conducted with an ozone concentration of 6.28 mg/L at the flow rate of 60 mL/min for different times. The results showed that ozone was an effective detoxification agent because of its powerful oxidative role. Thin-layer chromatography and liquid chromatography-quadrupole time-of-flight mass spectra were applied to confirm and identify the ozonolysis products of aflatoxin B(1). A total of 13 products were identified, and 6 of them were main products. The structural identification of these products provided effective information for understanding the ozonolysis pathway of aflatoxin B(1). Two ozonolysis pathways were proposed on the basis of the accurate mass and molecular formulas of these product ions. Nine ozonolysis products came from the first oxidative pathway based on the Criegee mechanism, and the other four products were produced from the second pathway based on the oxidative and electrophilic reactions of ozone. According to the toxicity mechanism of aflatoxin B(1) to animals, the toxicity of aflatoxin B(1) was significantly reduced because of the disappearance of the double bond on the terminal furan ring or the lactone moiety on the benzene ring.