<p>Gastrointestinal organoids, as advanced three-dimensional (3D) in vitro culture models, have revolutionized our understanding of organ development, disease pathophysiology, and therapeutic responses. However, their clinical translation is largely hampered by a reliance on Matrigel. Derived from mouse sarcoma, Matrigel presents inherent limitations, including an undefined composition, significant batch-to-batch variability, potential biosafety risks, and an inability to faithfully mimic the native tissue microenvironment. These limitations severely restrict the application of organoids in regenerative medicine and precision medicine. To overcome these barriers, tissue-specific decellularized extracellular matrix (dECM) has emerged as a superior alternative. Decellularized ECM is derived from native tissues through physical, chemical, or enzymatic removal of cellular components while preserving the intact 3D architecture, tissue-specific protein composition (matrisome), and bioactive signaling molecules of the native extracellular matrix. By preserving the complex 3D architecture, tissue-specific biochemical composition, and critical biological signals of the native ECM, dECM provides a more biomimetic niche for organoid culture. This review begins by examining the anatomical and functional characteristics of the gastrointestinal tract, establishing the physiological context for organoid modeling. This review first outlines the limitations of traditional matrices and establishes the role of the ECM as an active regulator of the stem cell niche. We then detail the preparation process of gastrointestinal dECM, analyzing the selection and optimization of decellularization methods and their impact on the scaffold’s biological and mechanical properties. Critical analysis of dECM’s mechanical properties reveals that while decellularization may reduce initial matrix stiffness compared to native tissue, appropriate crosslinking and concentration optimization can restore biomechanical integrity while maintaining biochemical fidelity—addressing a key advantage over Matrigel, which lacks both tunability and tissue specificity. Furthermore, we highlight the superiority of dECM in promoting the formation, maturation, and function of gastrointestinal organoids (e.g., stomach, intestine, colon). The review also summarizes recent advances in the application of dECM-cultured gastrointestinal organoids in disease modeling (e.g., inflammatory bowel disease, gastrointestinal cancers, infectious diseases), high-throughput drug screening, personalized medicine, and regenerative medicine (e.g., tissue repair and transplantation). We compare dECM-based matrices with emerging polymer bioinks, highlighting that while synthetic materials offer greater control over mechanical properties, they cannot replicate the complex biochemical signaling and growth factor reservoirs inherent to native ECM—a limitation that current bioengineering approaches continue to address through hybrid formulations. Finally, we discuss current challenges in dECM technology—such as standardization, scalable production, and complexity reconstruction—and envision future directions involving its integration with emerging technologies like 3D bioprinting, microfluidic chips, and multicellular co-culture systems. Such integrated approaches will catalyze the development of more physiologically relevant and functionally robust in vitro gastrointestinal models, opening new avenues for basic research and clinical translation.</p>