A new electrochemical immunosensing protocol by coupling with a magneto-controlled flow-through microfluidic device was developed for the sensitive detection of alpha-fetoprotein (AFP) on magnetic beads (MB) using ferrocene derivative polymer nanospheres (FDNP) as the electroactive mediators. The immunosensing probe was prepared by covalent conjugation of monoclonal mouse anti-human AFP antibodies with magnetic beads, while the recognition element was constructed by means of immobilizing polyclonal rabbit anti-human AFP antibodies on the redox FDNP. Upon target AFP introduction, the sandwich-type immunoreaction was carried out between the immunosensing probe and the recognition element, and the formed immunocomplex was captured in the detection cell with an external magnet. Ferrocene polymer nanospheres synthesized by infinite coordination polymerization were utilized as the signal-generation tags during the electrochemical measurement. Under optimal conditions, the magneto-controlled flow-through immunosensing platform exhibited good electrochemical responses toward target AFP within a dynamic working range of 0.01–100 ng mL−1 and with a low detection limit of 5.7 pg mL−1. The nanoparticles-based sensing systems also gave good reproducibility, high specificity and long-term stability. Moreover, our strategy displayed well-matched accuracy for the analysis of human serum specimens relative to commercial Roche 2010 Electrochemiluminescence (ECL) Automated Analyzer.