The invention discloses a preparation method of large-layer spacing vanadium pentoxide. The preparation method comprises the following steps: mixing vanadium pentoxide and a primary amine intercalateaccording to a molar ratio of (1-2):1, adding anhydrous ethanol, stirring uniformly, then adding deionized water, continuing stirring, transferring a mixture to a polytetrafluoroethylene-lined stainless steel high-pressure reaction kettle after stirring uniformly, reacting for 120-168 h at 160-180 DEG C, cooling to room temperature, then centrifugally washing an obtained black product with absolute ethanol and hexane, and drying and grinding to obtain the large-layer spacing vanadium pentoxide. Long-chain organic primary alkylamine is intercalated into a space between vanadium pentoxide layersby a solvothermal method to enlarge the layer spacing, and the layer spacing of the vanadium pentoxide can be adjusted through change of the chain length of the organic matter. The large-layer spacing vanadium pentoxide has excellent physical and chemical properties and has an extremely broad application prospect.