Abstarct: In this study, an optical sensor for determination of glutathione has been developed. This optical sensor is prepared by immobilization of 1-(2-Thiazolylazo)-2-naphtol (TAN) on triacetylcellulose membrane and subsequently formation of palladium (II) complex with immobilized ligand. The response of sensor is baxsed on the reaction of glutathione with palladium (II)-TAN complex. The decrease of the absorbance of Pd-TAN complex in the presence of glutathione was measured spectrophotometrically at 660 nm and used as analytical signal. Effects of important variables on the sensitivity of sensor were studied and optimized. Under the optimum conditions and 10, 15, 20 and 25 min response times, calibration curve was linear in the range of 1.50 – 22.0 mg L-1 of glutathione and the 3σ limit of detection was found 1.30, 0.90, 0.74 and 0.59 mg L-1, respectively. The relative standard deviations for five replicate measurements of 2.50, 10.0 and 20.0 mg L-1 of glutathione in 15 min, was 8.80 %, 7.60 % and 3.60 %, respectively. Also, the proposed sensor shows sufficient repeatability and reproducibility. Finally, this method was successfully applied for determination of glutathione in real samples including water, human serum samples with satisfactory results.