To boost the operational performance of a non-aqueous DES electrolyte-based vanadium-iron redox flow battery (RFB), our previous work proposed a double-layer porous electrode spliced by carbon paper and graphite felt. However, this electrode’s architecture still needs to be further optimized under different operational conditions. Hence, this paper proposes a multi-layer artificial neural network (ANN) model to predict the relationship between vanadium-iron RFB’s performance and double-layer electrode structural characteristics. A training dataset of ANN is generated by three-dimensional finite-element numerical simulations of the galvanostatic discharging process. In addition, a genetic algorithm (GA) is coupled to an ANN regression training process for optimizing the model parameters to elevate the accuracy of ANN prediction. The novelty of this work lies in this modified optimal method of a double-layer electrode for non-aqueous RFB driven by a machine learning (ML) model coupled with GA. The comparative result shows that the ML model reaches a satisfactory predictive accuracy, and the mean square error of this model is lower than other popular ML regression models. Based on the known region of operating conditions, the obtained results prove that this well-trained ML algorithm can be used to estimate whether a double-layer electrode should be applied to a non-aqueous vanadium-iron RFB and determine an appropriate thickness ratio for this double-layer electrode.