Monitoring blood lactate concentration is important in clinical diagnosis and evaluation of fitness care. Electrochemical enzyme sensors are commonly used for the measurement of lactate owing to their simplicity and sensitivity. Lactate oxidases (LOxs) are widely used for the construction of lactate enzyme sensors, especially the LOx derived from Aerococcus viridans (AvLOx). To minimize interference from redox substances, most lactate sensors utilize artificial mediators. However, reports on the comparison of different mediators in lactate measurements are limited. In this study, the preferences of AvLOx for electron mediators were investigated electrochemically. We concluded that the charge of the mediator plays a significant role in determining its suitability as a mediator for AvLOx. The relatively uncharged 1-methoxy-5-methyl phenazinium methylsulfate (mPMS) was the preferred mediator for AvLOx, the strongly negative ferricyanide was a less preferential mediator, and the strongly positive hexaammineruthenium(III) was not utilized as a mediator by AvLOx. This suggests that the protein scaffold of AvLOx can almost completely block the access to the co-factor for very small electron acceptors, depending on their charge.