Abstract: Peanut oil is derived from Arachis hypogaea Linn., and it has great nutritional value due to its high contents of active unsaturated triacylglycerols. Structural identification of these active unsaturated triacylglycerols provides us the opportunities to understand the biological functions of peanut oil for human health. In this study, a method by coupling Patrenò-Büchi reaction with ambient nanoelectrospray ionization mass spectrometry (PB-nanoESI-MS) was established for the identification of unsaturated triacylglycerols in peanut oil, and benzophenone was used as photochemical reactant. The peanut oils were dissolved in methanol-chloroform (9∶1, V/V) containing 0.4 mg/L benzophenone, and the sample solution was siphoned into a nanospray tip and then irradiated by a 254 nm UV for approximately 1 min. The cycloaddition reaction of CC in polyunsaturated triglycerides with carbonyl group in benzophenone was carried out. After that, nanoESI-MS analysis was performed under ambient and open-air conditions, and the structures of unsaturated triglycerides in peanut oil were identified by high-resolution and tandem mass spectrometry. The use of benzophenone as PB reagent resulted in 182 u mass increase for PB product ions, of which was easily discriminated from its original ions in the MS spectrum. When collision-induced dissociation (CID) experiment was performed to the PB product ion, CC diagnostic ion pairs were produced. Due to a mass difference of 150 u between O and C₁₃H₁₀, two fragment ions with 150 u mass difference were observed in the MS/MS spectrum, which were the diagnostic ions and applied to identify CC locations. As a result, a total of 29 major unsaturated triglycerides are successfully identified, and peanut oil is rich in the structure of FA18∶1(Δ9), FA18∶1(Δ12), and FA18∶2(Δ9,12). All the experiment results demonstrated that the developed PB-nanoESI-MS method is simple, fast, accurate, and effective, being a promising prospect in the traceability analysis as well as function and quality evaluation for edible oils.