The transient build-up of L-lactate in tissue is associated with acidosis, as occurs during hypoxia, and traditionally viewed as deleterious1–3. However, recent studies contradict this view by demonstrating L-lactate's vital role as an intercellular and interorgan exchangeable fuel4. As one example, the astrocyte-to-neuron lactate shuttle (ANLS) hypothesis proposes that neurons import L-lactate from astrocytes via the extracellular space to produce adenosine triphosphate (ATP) and sustain heightened neuronal activity5,6. To address the validity of this and related hypotheses on the emerging roles of L-lactate in brain energetics6,7,8, we introduce R-eLACCO2.1, a red fluorescent extracellular L-lactate biosensor. R-eLACCO2.1 enables two-color imaging, is superior to a contemporary green fluorescent biosensor in terms of sensitivity as measured in in vivo images from mouse neocortex, and is an effective fluorescence lifetime-based biosensor. To highlight the potential insights gained from in vivo measurements with R-eLACCO2.1, we performed dual-color imaging from somatosensory cortex of actively locomoting mice. This enabled us to observe the interplay between neural activity, reported by intracellular GCaMP expression, and extracellular L-lactate. As the highest-performance tool in the suite of extracellular L-lactate biosensors, R-eLACCO2.1 is ideally suited to delimit the emerging roles of L-lactate in mammalian metabolism.