Membrane type matrix metalloproteinases (MT-MMPs) play important roles in malignant tumor progression through the degradation of the extracellular matrix and signal transduction. However, a member of the family, MT3-MMP, has attracted the least concern compared with other MT-MMPs. Here, a novel MT3-MMP-targeting peptide with high affinity and specificity has been developed by a phage-display peptide screening technology and multiple biophysics measurements, including single-molecule recognition force spectroscopy and isothermal titration calorimetry. The binding peptides are conjugated on the surface of CdSe/ZnS quantum dots (QDs) and consequently acted as a ligand that specifically targets MT3-MMP overexpressed tumor cells. The imaging nanoprobes used QDs as the photographic developer for optical imaging in vivo. The nanoprobes exhibited a desirable targeting effect and generated good biodistribution profiles for visualization and imaging of MT3-MMP overexpressed tumor. The peptide could be useful to evaluate the distribution and expression of MT3-MMP. Furthermore, the peptide-functionalized QDs show potential application for cancer diagnosis.