Here, we demonstrate for the first time a straightforward and non-destructive 3D surface enhanced Raman spectroscopy (SERS) imaging approach to track the cellular uptake and localisation of AuNPs functionalised with an anti-ERα (estrogen receptor alpha) antibody in MCF-7 ERα-positive human breast cancer cells under different conditions including temperature and dynamin inhibition. Here, ESR1 is linked to breast cancer.