Introduction: The characteristics of the tumor immune microenvironment (TIME) are closely related to immunotherapy. Breast cancer can benefit from immunotherapy, and its TIME is still unclear. Methods: We utilized mass cytometry to explore the immune cell heterogeneity in breast cancer. Double-negative T cells (DNTs) from healthy volunteers (HBs) were enriched in vitro. Flow cytometry was used to detect the cell surface receptors of cancer cells and DNT cells. The correlation between immune checkpoints and the abundance of immune cells or prognosis of breast cancer was analyzed by the TCGA database. The messenger RNA (mRNA) expression of functional genes was performed by quantitative real-time PCR. Results: We found that the frequencies of Granzyme B (GZMB)(+)CD8(+)T and GZMB(+)DNT cells in cancer tissues (CA) of breast cancer were lower than those in blood samples of patients (PB), and the frequencies of programmed cell death protein 1 (PD1)(+)CD8(+)T and PD1(+)DNT cells in CA were higher than those in PB. DNTs from HBs had a cytotoxic effect on MDA-MB-231. LAG3Ab could upregulate the mRNA expression of interferon gamma and perforin by increasing T-BET transcription to enhance the cytotoxicity of DNT cells in vitro. Conclusion: Our study revealed the suppressive status of TIME in breast cancer and supported DNT cells had the potential to be applied as a novel adoptive cell therapy for TNBC either alone or combined with LAG3Ab.