<p>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&#xa0;cm⁻¹ weak. Benefitting from the synergistic normalization by MBN at 2223&#xa0;cm⁻¹ in the cell silent region, this ratio-type readout signal (<i>I</i>₂₂₂₃/<i>I</i>₁₆₅₀) was resistant to the endogenous and exogenous interference, contributing to the reproducibility and stability. The experimental results showed that the nanoprobe was capable of detecting MMP-2 activity at concentrations ranging from 10 to 100&#xa0;ng/mL, and the limit of detection could be down to 0.715&#xa0;ng/mL. Importantly, it was successfully used to differentiate the breast cancer cells from the normal cells based on the MMP-2 activity, which could have a great potential in the fields of tumor biology and accurate disease diagnosis.</p> Graphical abstract <p></p>