Membrane type-1 matrix metalloproteinase (MT1-MMP) plays a crucial role in many physiological and pathological processes, especially in tumor invasion and metastasis. Bioimaging of this key molecule may find wide usage in various applications. MT-loop is a unique sequence of MT1-MMP and locates in the surface of the protein. In our previous studies, AF7p, an affinity peptide that targeting the MT-loop domain of MT1-MMP, was identified by screening a phage display (Ph.D.) peptide library. However, the target of AF7p is a synthetic sequence which lacked native conformation of the MT-loop region; thus, the binding affinity and specificity in reality may not be optimal. In this study, we considered the 3-dimensional (3-D) conformation of the MT-loop area in the MT1-MMP molecule and designed a novel strategy to screen the Ph.D. peptide library. The peptide we obtained showed a better binding affinity to WT-MT1-MMP than AF7p as observed through enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). The new peptide labeled and attached MT1-MMP expression cell lines HT1080 and did not show any toxicity to cells. Furthermore, for in vivo imaging, HT1080 tumor-bearing mice with higher MT1-MMP expression accumulated more Cy5.5-HS7 than mice with MT1-MMP low-expression cell lines A549 at tumor sites, and the half-life of HS7 was longer than that of AF7p, as confirmed by ex vivo imaging of the main organs. These results suggest the feasibility of using the subtraction biopanning strategy to screen the affinity peptide targeting MT-loop regions and HS7 is a superior probe for noninvasively imaging MT1-MMP expression in MT1-MMP-positive tumor models. It provides impetus for further studies to use HS7 in early diagnosis of tumors and in peptide-mediated drugs.