Near-infrared (NIR) fluorescence optical imaging is an emerging imaging technique for studying diseases at the molecular level. Optical imaging with a NIR emitting fluorophore for targeting tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The previous in vitro results demonstrated that the GX1 peptide, identified by phage-display technology, is a tumor vasculature endothelium-specific ligand. In this report, Cy5.5-conjugated GX1 peptide was evaluated in a subcutaneous U87MG glioblastoma xenograft model to investigate tumor-targeting efficacy. The in vitro flow cytometry results revealed dose-dependent binding of Cy5.5-GX1 peptide to U87MG glioma cells. In vivo optical imaging with the Cy5.5-GX1 probe exhibited rapid U87MG tumor targeting at 0.5 h p.i., and high tumor-to-background contrast at 4 h p.i. Tumor specificity of Cy5.5-GX1 was confirmed by effective blocking of tumor uptake in the presence of unlabeled GX1 peptide (20 mg/kg). Ex vivo imaging further confirmed in vivo imaging findings, and demonstrated that Cy5.5-GX1 has a tumor-to-muscle ratio (15.21 ± 0.84) at 24 h p.i. for the non-blocked group and significantly decreased ratio (6.95 ± 0.75) for the blocked group. In conclusion, our studies suggest that Cy5.5-GX1 is a promising molecular probe for optical imaging of tumor vasculature.