Lymphatic metastasis is an important prognostic indicator for cancer progression. It is therefore considerably meaningful to develop molecularly targeted imaging probes for noninvasive and accurate identification of metastatic lymph nodes (MLNs) at early stages of tumor metastasis. Herein, we report a novel matrix metalloproteinase-2 (MMP-2)-activatable probe constructed with a near-infrared dye (Cy5), a quencher (QSY21), and a tumor-targeting peptide cRGD covalently linked through a radionuclide (125I)-labeled peptide substrate for accurate detection of MLNs. Upon cleavage with activated MMP-2, the above probe emitted MMP-2 concentration-dependent near-infrared fluorescence, which allows sensitive and specific visualization of MLNs via both optical and single-photon emission computed tomography imaging techniques. We thus envision that this probe would serve as a useful tool for studying tumor-induced lymphangiogenesis.