Lactate sensing has high importance for metabolic disease diagnostics, food spoilage, sports medicine, or the construction of biofuel cell devices. Therefore, continuous lactate sensing devices which enable accurate detection should be developed. Here we present the overexpression and utilization of FMN-lactate dehydrogenase from Saccharomyces cerevisiae for oxygen-insensitive, continuous amperometric lactate biosensing. The developed sensors exhibit a high signal-to-noise ratio, low interference effect, and a wide range of linear responses using both direct and mediated electron transfer configurations. The thionine-based mediated electron transfer configuration was stable for 8 h of continuous activity and two weeks of periodic activity with storage at 4 °C. We further grafted the redox mediators on multiwall carbon nanotubes to lower the redox mediator leaching effect. The developed grafting technique improved the biosensor stability and allowed continuous operation for at least 20 h. Both the mediator-entrapped and the grafted bioanodes were further coupled with a bilirubin oxidase-based biocathode to construct a biofuel cell device. The various biofuel cells have generated a maximal power output of 110 µW/cm² under atmospheric conditions and 200 µW/cm² under oxygen saturation.