The invention provides a preparation method of an electrolyte separator for the vanadium battery. The preparation method comprises the following steps of 1) preparing quaternary ammonium silane; 2) preparing a polymer; and 3) preparing the separator. The invention also discloses the electrolyte separator, obtained by the preparation method of the electrolyte separator for the vanadium battery, forthe vanadium battery and the vanadium battery employing the electrolyte separator for the vanadium battery as an electrolyte separator. Compared with a traditional quaternary ammonium salt negative ion exchange membrane in the prior art, the electrolyte separator for the vanadium battery, prepared by the method has the advantages of lower cost, better mechanical property, better chemical stability and better thermal stability, more excellent vanadium blocking performance, higher ion conductivity and small deformation, and the application requirement of the electrolyte separator for the vanadium battery is met.