A sensitive and rapid approach to perform testosterone (T) competitive immunoassay by microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed. The assay is based on the competitive immunoreactions between T and N-(4-aminobutyl)-N-ethylisoluminol-labeled T (ABEI-T) with a limited amount of antibody (Ab), and the rapid electrophoretic separation of an equilibrated mixture of ABEI-T-Ab complex and free ABEI-T, followed by CL detection using horseradish peroxidase-catalyzed ABEI-H2O2 system. Free ABEI-T and the ABEI-T-Ab complex are well separated within 30?s under the assay conditions. The developed method could be used to determine T with good precision and a detection limit lower than 1.0?nM. This method was applied for the quantification of T in human serum. The results demonstrated that the current MCE-CL-based competitive immunoassay maybe served as an alternative tool for clinical analysis.