An homogeneous immunoassay based on microchip electrophoresis (MCE) separation and chemiluminescence (CL) detection was developed for the determination of cortisol. The proposed method deployed the competitive immunoreaction of cortisol and horseradish peroxidase-labeled cortisol (HRP-cortisol) with a limited amount of anti-cortisol mouse monoclonal antibody (Ab). Free and bound HRP-cortisol were then separated by MCE. The effect of various factors such as conditions for the CL reaction, MCE separation and incubation time for the immunoreactions were examined and optimized. Under the optimal assay conditions, the MCE separation was accomplished within 60 s.Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H 2 O 2 reaction. The linear range for cortisol detection was 9-900 nM, and the limit of detection was 4.2 nM (S/N ¼ 3). The suitability of the proposed method for the quantification of cortisol in human serum was demonstrated.