Compared with conventional aqueous electrolyte, due to unique merits of deep eutectic solvents including a wide electrochemical window, ease in preparation, low vapor pressure and low cost, they have been used as electrolyte in the non-aqueous flow battery. This work reports a reline deep eutectic solvent, which is prepared as the anolyte and catholyte of a vanadium-iron redox flow battery. Since the solubility of the vanadium and iron species in deep eutectic solvent has been improved, an increased specific capacity can be obtained, up to 54.6% compared with the aqueous electrolyte one. However, the viscosity of this electrolyte is much larger than that of aqueous electrolyte, which causes more pumping losses. With the increase of operating temperature, the viscosity experiences a huge drop. The open-circuit voltage of such an non-aqueous system begins near 0.9 V with a decrease rate of 0.005 V h−1, suggesting a better capacity retention ability than does the aqueous flow battery. The cycling performance of this redox flow battery is evaluated at a current density of 2.0 mA cm−2 under the temperature of 30 °C. The coulombic efficiency is up to 94.8%, while voltage efficiency and energy efficiency are just 68.3% and 64.7%, respectively. The low voltage efficiency stems from the sluggish redox kinetics of the vanadium ions, and large internal ohmic resistance. With an improved thermal stability, this vanadium-iron redox flow battery is preferred to be operated at elevated temperature, which helps to greatly reduce the ohmic loss and pumping loss, such that to increase the system energy efficiency.