ABSTRACTImmunotherapy has demonstrated groundbreaking clinical efficacy across diverse cancer types. Nevertheless, its therapeutic potential remains constrained, with only a small subset of patients achieving durable clinical responses. Emerging evidence highlights tertiary lymphoid structures (TLSs), ectopic lymphoid aggregates induced by chronic inflammation or malignancy, play critical roles in mediating positive responses to cancer immunotherapy. Currently, there is a notable absence of comprehensive systematic evaluations that synthesise both clinical and preclinical experimental data regarding the role of TLSs in enhancing cancer immunotherapy. In this review, we examined the composition of TLSs and elucidated how their heterogeneity influences cancer treatment outcomes, with implications for both clinical practice and translational research. Furthermore, we emphasised the role of T follicular helper cells–mediated positive selection of germinal centre B cells in driving high‐affinity antibody production. Additionally, we comprehensively analysed the effects of pharmacological agents, oncolytic viruses, adeno‐associated viruses and biomaterials on TLSs formation and highlighted their capacity to potentiate immune checkpoint inhibitor responses. Consequently, targeting TLSs is a promising strategy for enhancing the positive response to cancer immunotherapy.