 The Br2/Br− oxidation–reduction couple is employed as the positive electrolyte in some redox flow batteries, such as zinc–bromine, vanadium–bromine, and hydrogen–bromine batteries. However, the Br2 and Br− crossover behavior with the proton exchange membrane is detrimental in these flow batteries. To explore the mechanism of the Br2 and Br− crossover, a spectrophotometer and a special reagent were developed to detect the accumulative penetration of the Br2 and Br− in the negative electrolyte. In addition, a laboratory-designed quinone–bromine flow battery was developed to investigate the effect of Br− and Br2 crossover behavior on cell performance. What’s more, molecular dynamics simulations of Br− and Br2 passing through a membrane were performed to analyze Br− and Br2 crossover behavior. It can be concluded that Br2 crossover was the key factor contributing to the current inefficiency. The coulombic efficiencies increased from 72.5 to 82.5 to 90% as the current density increased from 0.025 to 0.075 A cm−2 which occurred due to Br species crossover. The cell performance factors are strongly related to the amount of Br2 crossover; hence, these factors will vary as the bulk aqueous phase Br species concentration changes.