Isatidis Folium, derived from the dried leaves of Isatis indigotica Fort., has been used for centuries as a traditional Chinese herb with antibacterial and antiviral properties. However, heterogeneity in cultivation conditions and climatic variations poses challenges to accurately and effectively evaluate its quality. Current quality control methods cannot provide a comprehensive and effective identification of herbal quality and preparation efficacy. This study aimed to investigate the impact of different environmental factors on the biosynthesis and accumulation of medicinal components and identify biomarker genes and functional proteins associated with abiotic stress responses of Isatis indigotica Fort. We proposed evaluating the quality of Isatidis Folium based on multi-component quantitative analysis and integrating transcriptomic, proteomic, and physiological indicators to elucidate the mechanisms of herbal quality variation. The results revealed that abiotic stress conditions significantly altered the levels of bioactive constituents, physiological indices, and specific genes and proteins. Notably, biological pathways such as porphyrin metabolism, photosynthesis, and carbon fixation by photosynthetic organisms were implicated in phototoxicity within the photosystem under abiotic stresses. Biological pathways related to indole metabolism, specifically phenylalanine, tyrosine, and tryptophan synthesis, tryptophan metabolism, and indole alkaloid synthesis, were recognized as critical regulatory networks modulating indole alkaloid content. Candidate biomarkers such as HemB, PsbB, RBS2, RIBA2, TRPA, and TRPB were identified as potential factors of quality deterioration under adverse conditions. Based on the integration of chemical analysis and multi-omics techniques, a new hierarchical quality control scenario for Isatidis Folium was finally proposed, providing a research foundation for the innovation-driven development of traditional Chinese medicine.