Quick and effective detection of biothiols in biological fluids has gained increasing attention due to its vital biological functions. In this paper, a novel reversible fluorescence chemosensor (L-Cu2+) based on a benzocoumarin-Cu2+ ensemble has been developed for the detection of biothiols (Cys, Hcy and GSH) in human urine. The chemosensing ensemble (L-Cu2+) contains a 2:1 stoichiometry structure between fluorescent ligand L and paramagnetic Cu2+. L was found to exclusively bond with Cu2+ ions accompanied with a dramatic fluorescence quenching maximum at 443 nm and an increase of an absorbance band centered at 378 nm. Then, the in situ generated fluorescence sluggish ensemble, L-Cu2+, was successfully used as a chemosensor for the detection of biothiols with a fluorescence "OFF-ON" response modality. Upon the addition of biothiols, the decomplexation of L-Cu2+ led to the liberation of the fluorescent ligand, L, resulting in the recovery of fluorescence and absorbance spectra. Studies revealed that L-Cu2+ possesses simple synthesis, excellent stability, high sensitivity, reliability at a broad pH range and desired renewability (at least 5 times). The practical application of L-Cu2+ was then demonstrated by the detection of biothiols in human urine sample.