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Breast cancer is a complex and heterogeneous disease with varying cellular, genetic, epigenetic, and molecular expressions. The detection of intratumor heterogeneity in breast cancer poses significant challenges due to its complex multifaceted characteristics, yet its identification is crucial for guiding effective treatment decisions and understanding the disease progression. Currently, there exists no method capable of capturing the full extent of breast tumor heterogeneity. In this study, the aim is to identify and characterize metabolic heterogeneity in breast tumors using immune cells and an ultrafast laser-fabricated Immuno Nano Sensor. Combining spectral markers from both Natural Killer (NK) and T cells, a machine-learning approach is implemented to distinguish cancer from healthy samples, identify primary versus metastatic tumors, and determine estrogen receptor (ER)/progesterone receptor (PR) status at the single-cell level. The platform successfully distinguished heterogeneous breast cancer samples from healthy individuals, achieving 97.8% sensitivity and 92.2% specificity, and accurately identified primary tumors from metastatic tumors. Characteristic spectral signatures allow for discrimination between ER/PR-positive and negative tumors with 97.5% sensitivity. This study demonstrates the potential of immune cell-based metabolic profiling in providing a comprehensive assessment of breast tumor heterogeneity and paving the way for minimally invasive liquid biopsy approaches in breast cancer diagnosis and management., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)