In this study, we introduce for the first time the use of non-destructive 3D SERS imaging for the investigation of the cellular uptake mechanisms of AuNPs functionalised with an anti-ERα (estrogen receptor alpha) antibody and BPE (1,2-bis(4-pyridyl)ethylene) Raman reporter (ERα-AuNPs), in breast cancer cells under different endocytosis pathway inhibition conditions. The gene discussed is ESR1; the disease is breast carcinoma.