Gold nanoparticles (AuNPs) are widely used in various applications such as cancer imaging and drugdelivery. The functionalisation of AuNPs has been shown to affect their cellular internalisation,accumulation and targeting efficiency. The mechanism of cellular uptake of functionalised AuNPs bydifferent cancer cells is not well understood. Therefore, a detailed understanding of the molecularprocesses is necessary to improve AuNPs for their selective uptake and fate in specific cellular systems.This knowledge can greatly help in designing nanotags with higher cellular uptake for more selective andspecific targeting capabilities with less off-target effects. Here, we demonstrate for thefirst timea straightforward and non-destructive 3D surface enhanced Raman spectroscopy (SERS) imagingapproach to track the cellular uptake and localisation of AuNPs functionalised with an anti-ERa(estrogenreceptor alpha) antibody in MCF-7 ERa-positive human breast cancer cells under different conditionsincluding temperature and dynamin inhibition. 3D SERS enabled information rich monitoring of theintracellular internalisation of the SERS nanotags. It was found that ERa-AuNPs were internalised byMCF-7 cells in a temperature-dependent manner suggesting an active endocytosis-dependentmechanism. 3D SERS cell mapping also indicated that the nanotags entered MCF-7 cells using dynamindependent endocytosis, since dynamin inhibition resulted in the SERS signal being obtained from, orclose to, the cell surface rather than inside the cells. Finally, ERa-AuNPs were found to enter MCF-7 cellsusing an ERareceptor-mediated endocytosis process. This study addresses the role of functionalisationof SERS nanotags in biological environments and highlights the benefits of using 3D SERS for theinvestigation of cellular uptake processes.