It appears that tumour-infiltrating neoantigen-reactive CD8 + T (Neo T) cells are the primary driver of immune responses to gastrointestinal cancer in patients. However, the conventional method is very time-consuming and complex for identifying Neo T cells and their corresponding T cell receptors (TCRs). By mapping neoantigen-reactive T cells from the single-cell transcriptomes of thousands of tumour-infiltrating lymphocytes, we developed a 26-gene machine learning model for the identification of neoantigen-reactive T cells. In both training and validation sets, the model performed admirably. We discovered that the majority of Neo T cells exhibited notable differences in the biological processes of amide-related signal pathways. The analysis of potential cell-to-cell interactions, in conjunction with spatial transcriptomic and multiplex immunohistochemistry data, has revealed that Neo T cells possess potent signalling molecules, including LTA, which can potentially engage with tumour cells within the tumour microenvironment, thereby exerting anti-tumour effects. By sequencing CD8 + T cells in tumour samples of patients undergoing neoadjuvant immunotherapy, we determined that the fraction of Neo T cells was significantly and positively linked with the clinical benefit and overall survival rate of patients. This method expedites the identification of neoantigen-reactive TCRs and the engineering of neoantigen-reactive T cells for therapy.