© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. Purpose: There is a clinical need for agents that target glioma cells for non-invasive and intraoperative imaging to guide therapeutic intervention and improve the prognosis of glioma. Matrix metalloproteinase (MMP)-14 is overexpressed in glioma with negligible expression in normal brain, presenting MMP-14 as an attractive biomarker for imaging glioma. In this study, we designed a peptide probe containing a near-infrared fluorescence (NIRF) dye/quencher pair, a positron emission tomography (PET) radionuclide, and a moiety with high affinity to MMP-14. This novel substrate-binding peptide allows dual modality imaging of glioma only after cleavage by MMP-14 to activate the quenched NIRF signal, enhancing probe specificity and imaging contrast. Methods: MMP-14 expression and activity in human glioma tissues and cells were measured in vitro by immunofluorescence and gel zymography. Cleavage of the novel substrate and substrate-binding peptides by glioma cells in vitro and glioma xenograft tumors in vivo was determined by NIRF imaging. Biodistribution of the radiolabeled MMP-14-binding peptide or substrate-binding peptide was determined in mice bearing orthotopic patient-derived xenograft (PDX) glioma tumors by PET imaging. Results: Glioma cells with MMP-14 activity showed activation and retention of NIRF signal from the cleaved peptides. Resected mouse brains with PDX glioma tumors showed tumor-to-background NIRF ratios of 7.6–11.1 at 4 h after i.v. injection of the peptides. PET/CT images showed localization of activity in orthotopic PDX tumors after i.v. injection of 68Ga-binding peptide or 64Cu-substrate-binding peptide; uptake of the radiolabeled peptides in tumors was significantly reduced (p < 0.05) by blocking with the non-labeled-binding peptide. PET and NIRF signals correlated linearly in the orthotopic PDX tumors. Immunohistochemistry showed co-localization of MMP-14 expression and NIRF signal in the resected tumors. Conclusions: The novel MMP-14 substrate-binding peptide enabled PET/NIRF imaging of glioma models in mice, warranting future image-guided resection studies with the probe in preclinical glioma models.