There is a great need to develop multifunctional nanoparticles (MFNPs) for cancer biomarker-based detection and highly selective therapeutic treatment simultaneously. Here we describe a facile approach of layer-by-layer-assembled MFNPs conjugated with monoclonal antibody anti-HER2, demonstrating the specific detection of breast cancer BT474 cells (biomarker HER2 positive) with a high signal-to-noise ratio. The MFNPs contain a well-defined core-shell structure of UCNP@Fe3O4@Au coated by poly(ethylene glycol) (PEG) and anti-HER2 antibody, displaying excellent dispersity in various aqueous solutions. This unique combination of nanoparticles and ligand molecules allows us to perform photothermal treatment (PTT) of the cancer cells, while simultaneously quantifying the distribution of MFNPs on a cancer cell surface induced by antigen-antibody binding events. An important finding is that cancer cells adjacent to each other or in physical proximity within micrometers may end up with different fates of survival or death in PTT. This dramatic difference is determined by the antigen-antibody binding events at the interface of MFNPs and cells because of tumor cell heterogeneity. Therefore, our experiments reveal a new scale of the highly localized feature of the photothermal effect at the single-cell level illuminated by a continuous-wave near-IR laser.