Abstract: A new amphoteric ion exchange membrane was prepared via an one-step method. The membrane was based on a polymer prepared with 2,5-bis(2,3,4,5,6-pentafluorophenyl)-1,3.4-exadiazole and 9,9-bis(4-hydroxyphenyl) fluorene as monomers. Functional groups were introduced into the polymer by nucleophilic aromatic substitution reaction to prepare the amphoteric ion exchange membrane. Active sites and reaction energy barrier were predicted using a density functional theory. The results show that simulation analysis and ¹H and ¹⁹F NMR spectra are consistent. The prepared membrane has a higher ionic conductivity and lower permeability of vanadium ion. The conductivity is 58.5 mS·cm^-1 at 70℃ and vanadium ion permeability coefficient is 1.56×10^-8 cm²·min^-1 which is two orders of magnitude less than the permeability of Nafion^® 117. This membrane can possibly be used for vanadium redox flow battery production.