Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells.