The invention discloses a preparation method of ultra-pure vanadium pentoxide. The preparation method comprises the following steps of: removing impurities in advance; transferring phosphorus and silicon in a vanadium-containing solution into a solid phase for removal; performing calcification and vanadium precipitation; transferring vanadium in a liquid phase into a calcium vanadate phase; leaving the impurities such as chromium, manganese, potassium and sodium in the liquid phase for removal; performing solubilization reaction of vanadium in the calcium vanadate into the liquid phase through ammonium bicarbonate and/or ammonium carbonate; leaving the calcium, phosphorus, silicon, iron and magnesium in the solid phase for removal; replacing residual trace cations of the liquid phase with ammonium ions by an ammonium type cation exchange system to realize deep purification; and performing cooling vanadium precipitation and deamination calcination to obtain vanadium pentoxide. In the invention, the process route is simple, the drug consumption is low, the vanadium pentoxide with purity of over 99.95% can be stably obtained, and the content of main impurities such as iron, chromium, manganese, silicon, phosphorus, calcium, sodium, magnesium, potassium and aluminum is all lower than 0.002%.