The Transforming Growth Factor-beta (TGF-β) signaling pathway is a master regulator of cellular processes, including proliferation, differentiation and apoptosis, with profound implications for tissue homeostasis and disease pathogenesis. This review delves into the mechanisms and applications of TGF-β signaling within organoids, three-dimensional (3D) cultures that mimic human tissue architecture and function. We systematically explore the multifaceted role of TGF-β in organoid initiation, growth, and differentiation, highlighting its influence on stem cell self-renewal and lineage specification. The interplay between TGF-β and other signaling pathways, such as Wnt/β-catenin and Notch, is critical for maintaining tissue homeostasis and regulating stem cell niches. Furthermore, we discuss the significance of TGF-β signaling in modeling diseases like cancer and fibrosis, where its dysregulation is often implicated. Modulating TGF-β signaling in organoids holds vast therapeutic potential, offering insights into disease pathogenesis, predicting drug responses, and developing personalized treatment strategies. As our understanding of TGF-β signaling and organoid technology advances, these systems hold promise for groundbreaking biomedical research, potentially revolutionizing regenerative medicine and personalized therapeutics.