In traditional Chinese Medicine, Zhimu-Chuanbeimu (ZC) is one of the most classical herb pairs used in the treatment of lung diseases such as asthma. This study aimed to elucidate its specific components and potential mechanisms involved in treating asthma by altering the immune microenvironment. A systematic analysis of the chemical substance and absorbent components were conducted using UHPLC-Q-TOF-MS. Subsequently, a targeted network pharmacology based on serum pharmacochemistry was employed to predict core targets and key active ingredients. By combining microarray data analysis and gene set enrichment analysis, key targets and immune environment changes were further clarified. Ultimately, the prediction mechanism was validated through molecular docking, molecular dynamic simulations, and in vivo experiment validation. Consequently, sixty-seven prototype substances were identified in the blood samples of normal rats. Through network pharmacology and microarray data analysis, CCND1 and ESR1 were selected as key targets. Gene set enrichment analysis indicated that the expression of CCND1 was correlated with T helper cells, while the expression of ESR1 was positively correlated with mast cells. Additionally, the molecular docking and molecular dynamic simulations effectively supported the binding affinity between puqienine C and markogenin with the binding pockets of CCND1 and ESR1. Finally, in vivo experiments revealed that ZC extract could improve airway hyperresponsiveness, pulmonary inflammatory infiltration, the expression of CCND1/ESR1 and Th1/Th2/Th17/Treg imbalance in OVA-induced asthma rats. In conclusion, this research uncovered the pharmacodynamic foundation of ZC and its potential multifaceted pharmacological impacts, indicating that its potential anti-asthma mechanism might involve the modulation of immune cells and associated inflammatory pathways. Moreover, it provided a benchmark for enhancing asthma treatment protocols.