Dysregulation of receptor tyrosine kinases (RTKs) has been shown to correlate with cancer cell proliferation and drug resistance. Thus, monitoring the activity of RTKs at a chemical level could provide new biomedical insights and methods to assess the drug efficacy. However, direct monitoring of kinase activity is challenging and most commonly relies on in vitro techniques such as Western blotting and ELISAs. Herein, we report the development of a gold nanoparticle-based surface-enhanced Raman scattering (SERS) sensor, which allows the real-time monitoring of tyrosine phosphorylation of a reporter peptide (Axltide) by the Axl enzyme. We demonstrate that our sensor shows strong signal localization, and we are able to detect tyrosine phosphorylation of the reporter peptide through chemical phosphorylation and enzymatically with similar peak changes to those observed in the spontaneous Raman spectra. Through monitoring the SERS spectrum, we can observe changes in phosphorylation in real time.