Monoamine oxidases have two functionally distinct but structurally similar isoforms (MAO-A and MAO-B). The ability to differentiate them by using fluorescence detection/imaging technology is of significant biological relevance, but highly challenging with available chemical tools. Herein, we report the first MAO-A-specific two-photon fluorogenic probe (F1), capable of selective imaging of endogenous MAO-A enzymatic activities from a variety of biological samples, including MAO-A-expressing neuronal SY-SY5Y cells, the brain of tumor-bearing mice and human Glioma tissues by using two-photon fluorescence microscopy (TPFM) with minimal cytotoxicity.