Surface plasmon coupled emission (SPCE) technique has attracted 120 60 increasing attention in biomolectilar interaction analysis and-ell imaging because of its high sensitivity, low detection volume and low fluorescence background. Typically, the working range of SPCE is limited at nanometers to an interface. For micrometer-scale samples, new SPCB properties are expected because of complex coupling modes. In this work, cells with different subregions labeled were studied using a SPCE spectroscopy system. Angular and p-polarized emission was observed for cell membrane, cytoplasm, and nucleus labeled with DiI Nile Red, and propidiuni iodide, respectively. The SPCE signals were always partially p-polarized, and the maximum emission angle did not shift, regardless of variations in emission wavelength, fluorophore distribution and stained layer thickness. Additionally, increased polarization and a broader angle distribution were also observed with an increase in sample thickness. We also investigated-the impact of metallic substrates on the SPCE properties of cells. Compared with Au and Ni substrates) Al substrates presented better polarization and angle distribution. Moreover, the real-time detection of the cell labeling process was achieved by monitoring SPCE intensity. These findings expand SPCE from a surface technique to a 3D method for investigating bulk targets beyond the nanoscale interfaces, providing a basis to apply this technique to study cell membrane fluidity and bianinlecule interactions inside the cell and to distinguish between cellsubregions.