Sulfonated poly(ether ether ketone) (C-SPEEK) membranes with pendent carboxylic acid groups are prepared and first investigated for vanadium flow battery (VFB) application. The introduction of carboxylic acid groups can effectively improve the ion conductivities of C-SPEEK membranes, while, keep their ion selectivities. The prepared C-SPEEK membranes exhibit excellent performance under VFB operating condition. VFB single cell assembled with C-SPEEK-50 membranes shows much higher energy efficiency (85% Vs 82%) and columbic efficiency (97.3% Vs 94.6%) than that assembled with Nafion 115. The membrane keeps a stable performance after more than 180 cycles charge–discharge test, showing good stability.