A kind of gel beads loaded with iron-carbon powder (Fe-C), Zoogloea sp. L2, and riboflavin (VB2) were prepared through cross-linking of sodium alginate (SA) to establish an immobilized bioreactor. The optimal ratio of SA beads was adjusted by orthogonal experiment. The change of oxidation-reduction potential (ORP) and the concentration of Fe2+ and Fe3+ showed that the addition of VB2 as a redox mediator can promote denitrification. Under the optimal conditions (carbon to nitrogen (C/N) ratio = 2.0, pH = 7.0, and hydraulic retention time (HRT) = 8 h), the nitrate removal efficiency (NRE) of bioreactor reached 98.48% (1.99 mg L-1h-1). Furthermore, Fourier transform infrared spectrometer (FTIR), Fluorescence excitation-emission matrix (EEM), X-ray diffraction (XRD), and gas chromatography (GC) analysis revealed that the immobilization and denitrification of the immobilized bioreactor were excellent. High throughput sequencing also showed that Zoogloea played a vital role in nitrate removal.