Acid doped polybenzimidazoles (PBI) membrane is a type of promising proton exchange membrane for vanadium redox flow battery (VRFB) applications and increasing the basicity of PBI is an effective way to develop the acid doped level and proton conductivity. While according to Grotthuss mechanism, the proton conduct in PBI membranes can occur from protonated guest molecules to neighbor non-protonated neighbor molecules. Therefore, the flexibility of the polymer also can influence the proton conductivity of PBI membranes. In order to investigate in VRFB system which factor is more obvious, in this article, three PBI compounds with different basicity and flexibility were synthesized and then used to prepare dense proton exchange membranes for VRFB applications. The physicochemical properties and single cell performances of these membranes were valued. The results indicated that in VRFB system, the most flexible OPBI membranes show the highest proton conductivity. The VRFB used OPBI membrane as proton exchange membrane exhibited excellent cell performance, the energy efficiency (EE) of OPBI membrane reached 81.87% at current density of 100 mA cm−2, which was obviously higher than that of a commercial Nafion 115 membrane (EE = 77.69%). The self-discharge time of the OPBI membrane was recorded to be as long as 487 h, obviously longer than Nafion 115 (56 h).