There has been growing interest in the performance of vanadium redox flow batteries (VRFBs) depending on the electrolyte temperature and flow rate. In this work, we have devised a single-cell test system with four reservoirs which can effectively control the temperature and flow rate of VRFB to investigate electrochemical properties during discharging in VRFB. The temperature has been set between 278K and 318K for the electrolytes composed of 1600mol/m3 V3+/V4+ with 4000mol/m3 H2SO4, while the flow rate of the electrolytes is in the range of 10–100mL/min. The exchange current density extracted by Tafel theory is expressed by Arrhenius-like equation and ranges between 38.83 and 49.07A/m2. Meanwhile, the electron transfer coefficient increases from 0.31 to 0.51 with increased temperature and flow rate. The area-specific resistance is found to decrease with increased temperature at the rate of 20.3mΩcm2/K. With these, the proposed analytical method successfully predicts the obtained experimental data with excellent accuracy. Our study offers the fundamental understandings of electrochemical properties of VRFB as well as can be applied to evaluate the VRFB energy storage system at the early conceptual design even without prototypes.