An electrochemical immunosensor for real-time determination of corticosteroids, a group of steroidal hormones, is reported. The sensor measures competitive binding of endogenous corticosteroid and a corticosteroid-peroxidase conjugate with antibodies, immobilized on a platinum electrode, by monitoring of peroxidase activity. The electrode is encased within a dialysate membrane, which separates the electrode environment from the sampled fluid, allowing corticosteroid to equilibrate across the membrane. This permits measurements to be made in vivo. The small size of the probe (350 μm D) allows implantation into tissue or circulatory systems. The electrode was viable for 200 to 400 sequential measurements and at least 48 h in vivo. Detection sensitivity is 0.2 to 0.6 μg/100 ml of cortisol or corticosterone. The results suggest the possibility of monitoring hormones in real-time within the blood or organ systems of conscious animals.