In this study, electrochemical impedance spectroscopy (EIS) is evaluated as a novel application in the biodiesel purification process. EIS is proposed as a tool to study the behavior of charge carriers produced from side reactions or dissociations of some impurities during the gravitational separation of glycerol dispersed in crude biodiesel after the transesterification process. In the present study, EIS combined with a simple capacitor of two parallel plates made of stainless steel ASTM 304 with an area of 7.0 cm2 and a gap between the plates of 0.8 mm is demonstrated to be a promising technique to monitor the biodiesel/glycerol separation as a function of time. From the analysis of the impedance data, collected during the separation process, it was found that the biodiesel resistance increases up to a maximum value and then decreases to a value which corresponds to the final equilibrium state. Considering that the maximum resistance represents the less contaminated biodiesel as a criterion, the proper average biodiesel/separation time of 4 h 14 min ± 1 h 11 min was determined. Another finding from the EIS results suggests that a growing absorbed film exists on the plate surface of the capacitor used as an impedance sensor.