In this work, a new colorimetric method for the determination of Glutathione (GSH) on the basis of stable free radical 2,2,6,6 − tetramethylpiperidine − 1 − oxyl (TEMPO) oxidation of 3,3′,5,5′−tetramethylbenzizine (TMB) via copper(II) acetylacetonate (Cu(acac)₂) catalysis was proposed. TEMPO was catalyzed by Cu(acac)₂ to produce TEMPO⁺, then TEMPO⁺ oxidized TMB to produce oxidized TMB (ox − TMB). The resulting ox − TMB showed blue and possessed a distinct absorption peak about 650 nm. Whereas, GSH prohibited the generation of ox − TMB through inhibiting TMB oxidation. As compared to the case that GSH was absent, significantly enhanced absorption was determined, and was proportional to GSH amount. On this basis, a qualitative and quantitative detection method of GSH with the naked eye and the microplate reader was achieved. The developed TEMPO − based method achieved GSH biosensing with improved sensitivity in a good specificity − manner. The limit of detection (LOD) was 90 μM via naked eye, and the microplate reader was 4.71 μM. And the stable free radical TEMPO possessed higher stability and lower toxicity than traditional oxidant of H₂O₂. Moreover, this TEMPO − based method achieved good results in the detection of GSH in human serums.