A chitosan/glutathione composite film combined with glucose oxidase was employed to fabricate an amperometric glucose biosensor.The enzyme electrode was characterized by cyclic voltammetry.The experimental results showed that，chitosan and glutathione composite film can assist in electron transfer，improve response electrode current.Using orthogonal table L9（34） experiment design，the best experimental conditions were obtained by visual analysis.Under the optimized conditions，the sensor has a good linear relationship with concentration of glucose solution in the range of 1-18 mmol/L，the detection limit is 1.3 mmol/L.Experiments showed that，this sensor has the advantages of fast response，good stability and selectivity,and is suitable for the determination of glucose in urine sample.