Abstract: The content of unsaturated fatty glyceride in edible oil is closely related with human health. Edible vegetable oil contains high content of unsaturated fatty glyceride, while animal oil has much saturated fatty glyceride. When the edible vegetable oil was used repeatedly, the content of unsaturated fatty glyceride will decrease and that of saturated fatty glyceride will increase, which will bring bad effect to human health, so it is of great importance and value to establish a rapid and effective method to measure the content change of the whole unsaturated fatty glyceride profile. In this study, a novel high-resolution mass spectrometry method in atmospheric pressure chemical ionization mode (APCI-HRMS) for direct analysis of fatty glyceride in edible oil was developed. The oil samples were simply diluted with trichloromethane-methanol solvent (2∶1, V/V), and then directly injected into mass spectrometer without any separation. So the analysis is able to be performed quite fast (1.5 min/per sample) in a high throughput. Intensity difference can be directly observed in fatty acid triglyceride and fatty acid diglyceride comparing edible oil with waste oil. To confirm the intensity difference, the decomposition ratio (intensity of fatty acid diglyceride/intensity of fatty acid triglyceride) and average degree of unsaturation (DBE, average DBE of all the detected fatty acid triglyceride and fatty acid diglyceride) were calculated in edible vegetable oil and waste oil. It was found that the decomposition ratio obviously increased, while average DBE sharply decreased in waste oil. It shows that waste oil has similar characters with those of animal oil. The result demonstrates that decomposition ratio and average DBE can be used as the indicators to differentiate edible oil and waste oil. Furthermore, data matrix of fatty acid triglyceride and fatty acid diglyceride were under an orthogonal partial least squares discriminant analysis (OPLS-DA). The analysis result shows that the edible oil and waste oil are well separated, the grouped result validates that the differentiation is convincing. By calculating the decomposition ratio and average DBE of samples adding different portion of waste oil into edible oil, the method was further validated. It demonstrates that the method can easily accurately and fast measure the content change of fatty glyceride in oil, and it has the potential to become a rapid and effective method to monitor the quality of edible oil.