<p>Radix et Rhizoma Rhei (DaHuang, DH), subjected to different processing procedures, always result in different therapeutic effects because the interactions of the compounds co-occur. Herein, a chromatographic method was established using immobilized β₂-adrenoceptor (β2-AR) as the stationary phase to separate bioactive compounds in crude and processed DH. The approaches involved in the expression of β₂-AR in the <i>Escherichia coli</i> system, the immobilization of the receptor onto the surface of the macro-porous silica gel by a one-step site-specific covalent method, parallel screening of the bioactive compounds in crude- and processed- DH extract by the immobilized β₂-AR column, identification of the bioactive compounds by mass spectrometry, evaluation of the binding interaction of the compound by injection amount-dependent method, and the potential binding mechanism of the interaction between the retained compound by molecular docking. Five compounds were identified as the bioactive compounds that specifically bind to the immobilized β₂-AR column in crude-DH, while three of them were from processed-DH. The association constants of the compounds to the receptor were calculated as (5.78 ± 0.25) ×10⁵ M^− 1 for chrysophanol-8-O-β-glucoside, (0.70 ± 0.02) ×10⁵ M^− 1 for emodin-8-O-glucoside, (12.72 ± 1.83)×10⁵ M^− 1 for aloe emodin, (1.34 ± 0.07)×10⁵ M^− 1 for emodin, and (2.96 ± 0.17)×10⁵ M^− 1 for physcion, respectively. These results, taken together, not only confirmed the feasibility of the immobilized receptor chromatography in revealing the pharmacodynamic material basis of the crude and processed drug, but also provided an alternative for illuminating the underlying mechanism of the pharmacological activities of the processed drug.</p><p></p>