In this paper, we present a novel direct detection method for the characteristic fragrant component 2-acetyl-1-pyrroline (2-AP) in rice by using ultrasound-assisted solvent extraction (UASE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). No tedious derivatization procedures and toxic solvents were involved in the sample preparation process. 2-AP was extracted with ethanol by UASE from rice samples, and remained good stability within 27 h. By direct injection to UPLC-MS/MS, accurate results were obtained through matrix-matched internal standard calibration. Under optimized conditions, good repeatability (2.8%), reproducibility (7.6%), and recoveries (83.9–108%) were achieved with the limit of detection down to 0.3 μg/kg. The results of 2-AP in 13 rice samples determined by the present method were in the range of 19.4–124.0 μg/kg. The sensitivity and reliability of the developed UASE UPLC-MS/MS method are comparable with previously published SPME GC–MS/MS methods or derivatization-based HPLC–MS/MS method. High analysis throughput, low laboriousness, excellent accuracies, and low toxic solvent consumption are the main characteristics of the newly presented method.