Paeoniflorin, the major component of the root of Paeonia lactiflora, was quantitatively analyzed using 1H-NMR spectrometry. The quantity of paeoniflorin was calculated by the ratio of the intensity of the signals (H-9, H-10, H-2", 6") to the aldehyde peak of the known amount of internal standard, 2,4,6-trihydroxybenzaldehyde. These results were compared with the conventional HPLC method. The contents of paeoniflorin in P. lactiflora, which were respectively calculated by H-9, H-10, H-2", 6" in the 1H-NMR spectra and HPLC, were determined 2.60 ± 0.07, 2.44 ± 0.09, 2.77 ± 0.12 and 2.46 ± 0.16%. The advantages of quantitative 1H-NMR analysis are that can be analyzed to identify and quantify, and no reference compounds required for calibration curves. Besides, it allows rapid and simple quantification for paeoniflorin with an analysis time for only 20 min without any preprocessing.