Tertiary lymphoid structures (TLSs) usually lead to significantly improved clinical benefits in immunotherapy but are rarely observed within native tumors. The current approaches are difficult in effectively inducing TLS formation, let alone fully exploiting its anticancer efficacy. Here, a biomimetic nanovesicle (ADU-S@M1) is constructed to target tumors and then to produce abundant activated antigen-presenting cells (APCs) in situ by polarizing the tumor-associated macrophages toward M1 phenotype and promoting dendritic cell maturation. These activated APCs effectively initiate the TLS de novo neogenesis by acting as lymphoid tissue inducer cells that secrete lymphotoxin α and tumor necrosis factor α while normalizing the intratumoral vasculatures. In addition, they induce robust in situ adaptive immune responses by presenting the antigens released from the M1 cell-destroyed tumors and transporting them to the nearby TLS. Therefore, the development of tumors in mice, especially immune-cold tumors, was efficiently prevented, providing a promising strategy for promoting cancer immunotherapy.