As a precursor to cancer metastasis, the matrix metalloproteinase (MMP) family can degrade almost all protein components of the extracellular matrix, disrupting the histological barrier and promoting tumor invasion. Therefore, the sensitive and reliable detection of MMP activity in the tumor microenvironment is of great importance for the diagnosis and prognosis of malignant tumors. Here, a ratiometric surface-enhanced Raman scattering (SERS) sensing strategy based on interference-free internal standard was proposed for the accurate quantification of MMP-2 activity. A plasmonic substrate with core-satellite structure was constructed by self-assembly of silver nanoparticles on the gold core, which provided excellent SERS enhancement due to the coupling interaction. Besides, rhodamine B (RhB)-labelled substrate peptides and 4-mercaptobenzonitrile (MBN) were used as the MMP-2 recognizer and internal standard, respectively. MMP-2 specifically cleaved the peptides in half, leaving the RhB molecule free and the Raman signal at 1650 cm