The core of Chinese medicine compound prescription is the synergistic effect between the components and the effect of the interaction between the components on the dissolution and absorption of the drug. As a classic Chinese herbal formula, the laxative effect of Dahuang-Gancao decoction (DGD) is mainly derived from the anthraquinones in rhubarb. However, these components may also trigger adverse reactions due to their potency. Licorice, as a moderating herb, can alleviate the harshness of rhubarb. In this study, ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) technique was used to identify 28 constituents in DGD, mainly including anthraquinones, licorice flavonoids, licorice saponins and other constituents. Moreover, the effect of licorice on the in vivo bioavailability of rhubarb after compatibility was investigated based on biopharmaceutics approach. The results showed that licorice and its fractions (licorice polysaccharides, licorice total saponins and licorice total flavonoids) promoted the stability and solubility of the active ingredients in rhubarb (Aloe-emodin-8-O-β-D-glucoside, Sennoside B, Sennoside A, Aloe-emodin, Rhein, Emodin, Chrysophanol and Physcion) in varying degrees and thereby improved their bioavailability in vivo. In addition, although there was no change in the biopharmaceutics classification of anthraquinone components, their pairing mainly resulted in increased solubility and decreased permeability. These findings provide a theoretical basis for elucidating the compatibility mechanism of the two.