Surface enhanced Raman scattering (SERS) as a labelling technique offers multiplexing, quantification, high sensitivity, photostability, and reproducibility. Immuno-SERS (iSERS) microscopy combines SERS technique and the immuno activity of antibodies. In this work, iSERS is employed for breast cancer detection on tissue sections and cell slides. To obtain a false-colour iSERS image with high contrast, two different SERS substrates were synthesized with significant SERS enhancement. SERS-labelled antibodies were added onto tissue/cell slides to localize the targeted protein. The feasibility of imaging human epidermal growth factor receptor 2 (HER2) expression was firstly demonstrated on breast formalin-fixed paraffin-embedded (FFPE) tissue sections. To acquire a fast and reproducible iSERS image, instrumental parameters were then systematically investigated on breast cancer cell slides (SkBr-3). Finally, the localization of six SERS conjugates on a single SkBr-3 cell and the identification of breast cancer cells in a cell mixture were successfully performed using Au/Au coresatellites-labelled antibodies, respectively.