Neuromodulators impose powerful control over brain function via their regulation of intracellular signaling through G-protein coupled receptors. In contrast to those of Gs and Gi pathways, in vivo imaging of the signaling events downstream of Gq-coupled receptors remains challenging. Here, we introduce CKAR3, a genetically encoded fluorescence lifetime sensor that reports the activity of protein kinase C (PKC), a major downstream effector of the Gq pathway. CKAR3 exhibits a lifetime dynamic range 5-fold larger than any existing PKC sensor. It specifically detects PKC phosphorylation with seconds kinetics without perturbing neuronal functions. In vivo two-photon lifetime imaging of CKAR3 reveals tonic PKC activity in cortical neurons. Animal locomotion elicits robust PKC activity in sparse neuronal ensembles in the motor cortex. Both basal and locomotion-elicited PKC activities are in part mediated by muscarinic acetylcholine receptors. Overall, CKAR3 enables interrogation of Gq signaling dynamics mediated by PKC in behaving animals.