An innovative magnetic immunoassay was developed for the voltammetric detection of carbohydrate antigen-125 (CA-125) on a home-made microfluidic device including a multisyringe pump, selection valve and magneto-controlled detection cell. Two kinds of biofunctionalized nanostructures including anti-CA-125 capture antibody-conjugated magnetic beads and anti-CA-125 detection antibody-labeled silver-polypyrrole (Ag-PPy) nanohybrids were utilized for a sandwiched immunoreaction in the presence of CA-125. With the help of an external magnet, the formed magnetic immunocomplexes were attached to the sensing interface to activate the electrical contact between Ag-PPy nanohybrids and the base electrode, thus resulting in the switching on of the sensor circuit for the generation of voltammetric signals thanks to electroactive Ag-PPy nanohybrids. Compared to silver nanoparticles (AgNPs) alone, improved analytical properties were acquired with Ag-PPy nanohybrids. Under the optimal conditions, the currents depended on the concentrations of target CA-125, and exhibited a linear relationship within the ranges of 0.001-300 U mL-1 at a detection limit of 7.6 mU mL-1. For the determination of CA-125, the magnetic immunoassay had acceptable reproducibility, high specificity against other biomarkers and long-term storage stability. Moreover, good accuracy was obtained for the CA-125 detection in human serum samples with the developed voltammetric immunoassay relative to commercial enzyme-linked immunosorbent assay (ELISA). Importantly, the magneto-controlled immunosensing interface could be repeatedly used via detaching/attaching the external magnet.