Matrix metalloproteinases (MMPs) are endopeptidases responsible for degrading the extracellular matrix (ECM) and remodeling tissue in both physiological and pathological processes. MMP2 and membrane-type 1 MMP (MT1-MMP) have been associated with tumor invasion, metastasis and angiogenesis; therefore, a molecular imaging strategy assessing their activity may help to predict the malignancy of tumors. Here, we established a novel method of specifically tagging the surface of MMP2- and MT1-MMP-positive cells, and applied it to the development of an optical imaging probe. We constructed a protein-based probe composed of a glutathione-S-transferase (GST)-tag (Inhibitory [I]-domain), a polypeptide as a specific substrate for both MMP2 and MT1-MMP (Cleaved [C]-domain), a transmembrane domain of the epidermal growth factor receptor (Transmembrane [TM]-domain), and DsRed2 (Fluorescent [F]-domain). In vitro experiments clearly demonstrated that, after the probe was cleaved at the C-domain by the MMPs, the resultant TM–F-domain was inserted into the cellular membrane. Optical imaging experiments in vivo demonstrated that the probe was cleaved and specifically remained in tumor xenografts in a MMP-dependent manner. These results indicate that the release of the I–C-domain through the proteolytic cleavage of the C-domain by MMP2 and MT1-MMP triggers the tagging of cellular membranes with the TM–F-domain. The present feasibility study opens the door to the development of a novel imaging probe for tumor malignancy using positron emission tomography as well as an optical imaging device.