Changes in the intestinal immune micro-environment of the gastrointestinal tract are indispensable in the occurrence and development of gastrointestinal cancer. Tertiary lymphoid structure (TLS) is an immune cell aggregation structure found around gastrointestinal cancer in recent years. More and more research proves that tertiary lymphoid structure plays a key biological role and clinical value in disease progression, patient prognosis, and adjuvant treatment. This review aims to explore the research progress, biological significance, and potential clinical applications of TLSs in gastrointestinal tumors. The formation, development, and interaction of TLSs with tumor microenvironment have been reviewed and analyzed in recent years. Meanwhile, this review not only evaluates the clinical value of TLSs as prognostic biomarkers and predictors of treatment response but also explores their role in guiding the formulation of immunotherapy strategies for gastrointestinal tumors. In addition, this review points out the main problems in the current research of TLSs and looks forward to their future development, especially their broad application prospects in the diagnosis, treatment, and prognostic evaluation of gastrointestinal tumors.