Bifunctional redox flow batteries (BRFB) possess functions of both electricity storage and electrochemical preparation, having the potential for increasing the electrical energy utilization. A V(III)/V(II)–glyoxal(O2) system has been developed. Separators of the BRFB play a key role in BRFB performance. A Nafion solution was sprayed on a gas diffusion layer (GDL) at the Nafion loading of 2mgcm−2, and the GDL was then hot-pressed onto a Nafion115 cation exchange membrane, obtaining a modified separator. This separator not only prevents the crossover of vanadium but also has favorable conductivity, obtaining optimal charge and organic electro-synthesis performance of the BRFB. The effects of the concentrations of glyoxal and HCl on the performance of BRFB were also investigated. It is shown that the optimal concentration of glyoxal and HCl should be 1.2 and 3M, respectively. As a result, the current efficiency of organic electro-synthesis is further increased. An acceptable discharge performance is achieved for a period exceeding 20h at the current density of 20mAcm−2. The average discharge voltage of 0.73V and the coulombic efficiency of 66% are obtained. It is demonstrated that the principle of the BRFB is feasible. However, further experiments are needed to improve the performance.