Therapeutic target for over-expressed ${alpha}_v{eta}_3$ integrins in angiogenic endothelial cells and tumors is one of the promising approaches for cancer imaging and therapy. In the previous study, we demonstrated that heparin-lithocholic acid functionalized with cyclic RGDyK (cRGD-HL) had potent angiogenesis inhibition and tumor regression effects. The aim of this study is to validate the targeting property and specificity of cRGD-HL to ${alpha}_v{eta}_3$ integrin-expressing endothelial cells and tumor tissues by Cy5.5-labeled cRGDyK (RGD-Cy5.5) as ${alpha}_v{eta}_3$ integrin imaging agent and near-infrared fluorescence (NIRF) imaging systems. In this study, we demonstrated that cRGD-HL markedly inhibited the binding of fluorescein-labeled ${alpha}_v{eta}_3$ antibody to ${alpha}_v{eta}_3$ integrin- expressing endothelial cells when compared to nonfunctionalized heparin derivatives. Furthermore, in vivo NIRF images showed that cRGD-HL could decrease the NIRF signal intensities in both ${alpha}_v{eta}_3$ integrin-positive tumor (U87 MG) and ${alpha}_v{eta}_3$ integrin-negative tumor (SCC7) more effectively than non-functionalized heparin derivatives could. Therefore, with the help of ${alpha}_v{eta}_3$ integrin imaging agent and NIRF imaging systems, we verified that the functionalized cRGD-HL has much stronger tumor targeting property and specificity against ${alpha}_v{eta}_3$ integrin-expressing endothelial cells and tumors than non-functionalized heparin derivatives. Also, we believe that cRGD-HL will be useful and give affirmative outcomes for the treatment of angiogenesis-related diseases.