Shaoyao-Gancao-Fuzi decoction, a famous traditional Chinese medicine prescription and composed of the roots of Paeonia lactiflora Pall., Paeoniaceae, Glycyrrhiza glabra L., Fabaceae, and Aconitum carmichaelii Debx., Ranunculaceae, exhibits an anti-inflammatory and analgesic effects. However, pharmacokinetic studies of its potential active ingredients have rarely been studied. The aim of present study was to characterize the pharmacokinetic properties of albiflorin, paeoniflorin, benzoylmesaconine, liquiritin, liquiritigenin, glycyrrhizic acid, and glycyrrhetinic acid after administration of the Shaoyao-Gancao-Fuzi decoction in rat. The plasma concentrations of the seven active components were analyzed by liquid chromatography coupled to mass spectrometry with a good accuracy (87.62–109.71%), precision (2.96–11.38%), extract recovery (85.79–103.68%), matrix effect (90.38–107.77%), and stability (86.66–112.75%). As results, albiflorin, paeoniflorin, and benzoylmesaconine were rapidly absorbed with Tmax < 1 h and slowly eliminated with mean half-life of 6.22–7.11 h. Glycyrrhetinic acid exhibited a high bioavailability.