Receptor change induced by ligand binding is a new issue to face in the field of targeted delivery. Receptor clustering, the main pattern of receptor changes, decreases the affinity between ligand and receptor due to the redistribution of receptor position. In an attempt to respond to such challenge, we designed and constructed three RGD-modified nanocarriers with different ligand intervals: stealth liposomes modified with the monomeric RGD (moRGD-LP), dimeric RGD (diRGD-LP) and a special dimeric RGD with a linker between two cyclic RGD motifs (P-diRGD-LP). The αvβ3-positive and -negative tumor cells (Melanoma B16 and MCF-7) were used as the cell models. As a result, P-diRGD-LP demonstrated strongest interaction with B16 cells in surface plasmon resonance study and highest cellular uptake in B16 cells in real-time confocal analysis. The enhanced endocytosis of P-diRGD-LP was found to be αvβ3-mediated and P-diRGD-LP increased the involvement of the clathrin-dependent pathway. Importantly, P-diRGD-LP demonstrated the best targeting effect in B16-tumor bearing mice in both in vivo and ex vivo near-infrared fluorescent images, about 2.4-fold that of moRGD-LP and 2.8-fold that of diRGD-LP at 3 h. Further, we validated integrin αvβ3 clustering on B16 cells via a single-molecule imaging by a total internal reflection fluorescence microscopy. Finally, the 3D models of αvβ3 clustering suggested a receptor interval within 41.916-65.779 Å, while the molecular computation revealed an RGD ligand interval of 20.944 Å, 42.753 Å and 78.196 Å for diRGD-LP, P-diRGD-LP and moRGD-LP, respectively, confirming the best matching between clustered αvβ3 and P-diRGD-LP. In conclusion, P-diRGD-LP could achieve higher targeting to αvβ3-positive tumor via the enhanced interaction based on the better ligand-receptor compatibility. The design of targeted nanocarriers against receptor clustering might provide new insight into the nanotechnology-based anticancer therapy.