Current intravital microscopy techniques visualize tauopathy with high-resolution, but have a small field-of-view and depth-of-focus. Herein, we report a transcranial detection of tauopathy over the entire cortex of P301L tauopathy mice using large-field multifocal illumination (LMI) fluorescence microscopy technique and luminescent conjugated oligothiophenes. In vitro assays revealed that fluorescent ligand h-FTAA is optimal for in vivo tau imaging, which was confirmed by observing elevated probe retention in the cortex of P301L mice compared to non-transgenic littermates. Immunohistochemical staining further verified the specificity of h-FTAA to detect tauopathy in P301L mice. The new imaging platform can be leveraged in pre-clinical mechanistic studies of tau spreading and clearance as well as longitudinal monitoring of tau targeting therapeutics.