Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are three major biothiols, which play key roles in various biological systems. Accordingly, the development of imaging probes has been actively studied. We report a new pyrene derivative 1, which showed large fluorescence quenching with Cu(2+) at pH 7.4. The ensemble 1-Cu(2+) was applied to detect biothiols. Among the various amino acids, GSH, Cys, and Hcy induced distinct turn-on fluorescence changes. The 1-Cu(2+) ensemble was further applied for GSH detection in living cells.