Abstract: Calycosin, a pivotal isoflavonoid active constituent derived from Astragalus membranaceus, is designated as a key marker compound for the quality assessment of Astragalus and its products in the Pharmacopoeia of the People’s Republic of China (2020 Edition). It exhibits a broad spectrum of pharmacological activities and holds significant potential for clinical application. This article systematically reviews the research progress on calycosin. In terms of extraction, isolation, and purification, techniques such as flash extraction and hydrolytic extraction enable efficient enrichment of the compound, whereas methods such as macroporous adsorption resin and high-speed counter-current chromatography allow for high-purity preparation. The biosynthetic pathways of calycosin encompass the phenylpropanoid pathway in planta, the chemical “one-pot” method in vitro, and synthesis via microbial cell factories, offering diverse strategies for large-scale production. Structural derivatization, particularly through modification of the 7- and 3'-hydroxyl groups, significantly enhances its solubility and antitumor activity. With respect to pharmacological mechanisms, calycosin exerts multi-pathway and low-toxicity effects in diseases such as inflammation, cancer, and neural injury by modulating multiple signaling pathways, including NF-κB, PI3K/AKT, and MAPK. Furthermore, pharmacokinetic studies indicate that its absorption depends on deglycosylation, that it undergoes substantial hepatic first-pass metabolism, and that its tissue distribution is organ-specific. Safety evaluations suggest low toxicity at therapeutic concentrations. This review aims to clarify the core issues concerning the translation of basic research on calycosin into clinical practice, thereby providing a theoretical foundation for subsequent development.