Layer-by-layer V2O5 structures self-assembly by quadrate sheets like “multilayer cake” were synthesized. Carbon spheres is as the structure-directing reagent like adhesive to guide the formation of layer-by-layer structures. UV–vis spectrum shows two major absorption bands at about 340 and 478 nm and PL spectrum exhibits the emission peak at 545 nm for V2O5 layer-by-layer structures. The electrochemical properties of layer-by-layer V2O5 structures are significantly improved in organic electrolyte.