Accumulating evidence suggests that molecular dynamics at nanometer scale is crucial for brain functions and disorders. Single-molecule fluorescence imaging is a super-resolution live imaging method that enables direct tracking of movement of individual molecules. However, conventional single-molecule imaging has been applicable only to dissociated cells on coverslips due to technical limitations, preventing the analysis of events that occur only in the intact brain tissue. In this study, we set out to develop a method for single-molecule imaging within brain slices and the brain in vivo. We developed and employed a novel chemical tag technology named De-QODE. This technology consists of a small-molecular QODE probe and DeQODE protein tag. Non-fluorescent QODE becomes highly fluorescent upon reversible binding to DeQODE. These properties allow us to lower background and avoid photobleaching even in light-scattering tissue samples. We succeeded in continuous and high-density tracking of QODE molecules activated by membrane-tagged DeQODE in pyramidal neurons deep within acute cortical slices. This result indicates that our De-QODE-based method is highly promising to realize the pharmacology based on single-molecule dynamics.