A novel Fe3O4@Cu/PVA biomaterial as a new adsorbent and bacterial cell immobilized carrier was synthesized in this work. The structure and morphology were characterized by SEM. Effects of factors on Mn(II)-based autotrophic denitrification were investigated in a moving bed biofilm reactor (MBBR). The results indicate that the highest nitrate removal and Mn(II) oxidation efficiency occurred under the conditions of initial Mn(II) concentration of 80 mg·L−1, HRT of 10 h and pH 7. Meteorological chromatography analysis showed that N2 was produced as an end-product, and that gas compositions were different depending on the concentration of Mn(II) in the MBBR. The community diversity in the MBBR was markedly influenced by the concentration of Mn(II) and Pseudomonas sp. H-117 played a primary role in the process of nitrate removal and Mn(II) oxidation.