A fluorescent probe for glutathione (GSH) detection was developed. Our study indicates a possible mechanism which couples a conjugate addition and micelle-catalyzed large membered ring formation/elimination sequence. This method enables excellent selectivity towards GSH over other biological thiols such as cysteine (Cys) and homocysteine (Hcy). The proposed method is precise with a relative standard deviation (R.S.D) lower than 6% (n = 3) and has been successfully applied to determine GSH in human plasma with recoveries between 99.2% and 102.3%.