A novel reusable amperometric immunosensor was developed for the determination of cortisol by co-immobilizing horseradish peroxidase (POD) and cortisol-antibody on a chemically activated affinity membrane which is mounted over the tip of an oxygen electrode. The enzymatic electrocatalytic current response to the respective substrate is inhibited by the association of the antigen to the co-immobilized antibody. The sensor can be regenerated to facilitate several assays by washing with dilute hydrochloric acid solution. The advantages of the sensor include rapid-response, simple analysis methodology and high selectivity. The calibration curve for cortisol is linear in the concentration range of 1 × 10−7– 1 × 10−5M. The optimal conditions of immobilization and pH have been studied. The inhibition of enzymatic activity was confirmed by luminescence measurement utilizing the luminol- H2O2-POD system.