Tertiary lymphoid structures (TLSs) are ectopic lymphocyte aggregation structures found in the tumor microenvironment (TME). Emerging evidence shows that TLSs are significantly correlated with the progression of gastrointestinal tumors, patients' prognosis, and the efficacy of adjuvant therapy. Besides, there are still some immunosuppressive factors in the TLSs that may affect the anti-tumor responses of TLSs, including negative regulators of anti-tumor immune responses, the immune checkpoint molecules, and inappropriate tumor metabolism. Therefore, a more comprehensive understanding of TLSs' responses in gastrointestinal tumors is essential to fully understand how TLSs can fully exert their anti-tumor responses. In addition, targeting TLSs with immune checkpoint inhibitors and vaccines to establish mature TLSs is currently being developed to reprogram the TME, further benefiting cancer immunotherapies. This review summarizes recent findings on the formation of TLSs, the mechanisms of their anti-tumor immune responses, and the association between therapeutic strategies and TLSs, providing a novel perspective on tumor-associated TLSs in gastrointestinal tumors.