<div><p>Breast cancer is one of the most frequent and aggressive primary tumors among women of all races. Matrix metalloproteinase (MMPs), a family of zinc- and calcium-dependent secreted or membrane anchored endopeptidases, is overexpressed in varieties of diseases including breast cancer. Therefore, noninvasive visualization and quantification of MMP <i>in vivo</i> are of great interest in basic research and clinical application for breast cancer early diagnosis. Herein, we developed a <sup>99m</sup>Tc labeled membrane type I matrix metalloproteinase (MT1-MMP) specific binding peptide, [<sup>99m</sup>Tc]-(HYNIC-AF7p)(tricine)(TPPTS), for <i>in vivo</i> detection of MDA-MB-231 breast tumor by single photon emission computed tomography (SPECT). [<sup>99m</sup>Tc]-(HYNIC-AF7p)(tricine)(TPPTS) demonstrated nice biostability and high MT1-MMP binding affinity <i>in vitro</i> and <i>in vivo</i>. Tumor-to-muscle ratio was found to reach to the highest (4.17±0.49) at 2 hour after intravenously administration of [<sup>99m</sup>Tc]-(HYNIC-AF7P)(tricine)(TPPTS) into MDA-MB-231 tumor bearing mice. Overall, [<sup>99m</sup>Tc]-(HYNIC-AF7P)(tricine)(TPPTS) demonstrated great potential for MT1-MMP targeted detection <i>in vivo</i> and it would be a promising molecular imaging probe that are probably beneficial to breast cancer early diagnoses.</p></div