Pathological angiogenesis is crucial in tumor growth, invasion and metastasis. Previous studies demonstrated that the vascular endothelial growth inhibitor (VEGI), a member of the tumor necrosis factor superfamily, can be used as a potent endogenous inhibitor of tumor angiogenesis. Molecular probes containing the asparagine-glycine-arginine (NGR) sequence can specifically bind to CD13 receptor which is overexpressed on neovasculature and several tumor cells. Near-infrared fluorescence (NIRF) optical imaging 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 aim of this study was to develop a new NIRF imaging probe on the basis of an NGR-VEGI protein for the visualization of tumor vasculature. The NGR-VEGI fusion protein was prepared from prokaryotic expression, and its function was characterized in vitro. The NGR-VEGI protein was then labeled with a Cy5.5 fluorophore to afford Cy5.5-NGR-VEGI probe. Using the NIRF imaging technique, we visualized and quantified the specific delivery of Cy5.5-NGR-VEGI protein to subcutaneous HT-1080 fibrosarcoma tumors in mouse xenografts. The Cy5.5-NGR-VEGI probe exhibited rapid HT-1080 tumor targeting, and highest tumor-to-background contrast at 8 h post-injection (pi). Tumor specificity of Cy5.5-NGR-VEGI was confirmed by effective blocking of tumor uptake in the presence of unlabeled NGR-VEGI (20 mg/kg). Ex vivo NIRF imaging further confirmed in vivo imaging findings, demonstrating that Cy5.5-NGR-VEGI displayed an excellent tumor-to-muscle ratio (18.93 ± 2.88) at 8 h pi for the non-blocking group and significantly reduced ratio (4.92 ± 0.75) for the blocking group. In conclusion, Cy5.5-NGR-VEGI provided highly sensitive, target-specific, and longitudinal imaging of HT-1080 tumors. As a novel theranostic protein, Cy5.5-NGR-VEGI has the potential to improve cancer treatment by targeting tumor vasculature.