Abstract: This study compared the chemical composition differences among various root tissues of Astragalus membranaceus through optimization of histochemical localization methods, thereby providing some reference for targeted extraction and enrichment of bioactive components. The chloral hydrate permeabilization technique was enhanced with sodium nitrite-aluminum nitrate-sodium hydroxide reagent for flavonoid staining. Comparative analyses of total saponins, total flavonoids, and astragaloside IV content in xylem, phloem, and periderm tissues were performed using ultraviolet-visible spectroscopy (UV-Vis) and high-performance liquid chromatography (HPLC). By optimizing staining protocols, interference from natural pigmentation was successfully eliminated, thereby enhancing the visualization of tissue-specific chemical distribution. Quantitative analysis revealed that the phloem contained the highest total saponins (6.92%), while the periderm exhibited peak total flavonoids (0.876%) and exclusive enrichment of astragaloside IV (0.850%). The refined histochemical localization method enables precise characterization of phytochemical distribution across root tissues, which can offer guidance for selective extraction and enrichment strategies, and comprehensively promote the utilization and development of Astragalus membranaceus resources.