Spectrophotometry is a general method used in enzymatic synthesis of glutathione（GSH） for tracking detection of reaction process.However，-SH from cysteine（Cys） could significantly interfere the results of GSH analysis.By multiple interference experiments and that of data processing and principle analysis，an anti-interference compensation method for elimination of interference caused by the reaction of alloxan and Cys was established.Anti-interference mechanism was set by introducing interference coefficient and compensation coefficient，building interference model and compensation algorithm.The absorbance and derivative of unknown sample were detected at 320 nm，then the data detected were taken into the flow chart of anti-interference compensation，the interference ratio and compensation coefficient were obtained，and the contents of GSH and Cys could be calculated.The detection results of new method showed that when the concentration of Cys in reaction liquid was not more than 4 times of that of GSH，the recoveries of GSH and Cys were in the ranges of 95%-100% and 78%-120%,respectively.The disturbance of Cys to the detection results could be eliminated.The standard equation for GSH concentration and absorbance is y=3.062 9x+0.000 1，r2=0.999 9.And compared with alloxan method，it has a significant enhancement in the anti-interference characteristics for detection of the contents of GSH and semi-quantitation of Cys on the basis of multiple relationships.It has a practical application value to the quantitative detection of GSH with non-separating cysteine in enzymatic synthesis of GSH.