An immobilized biological filter (IBF) using Fe(II) and Mn(II) as mixed electron donors was evaluated for nitrate removal in groundwater. Results of the single factor experiments of strain SZ28 under the conditions of electron donor:electron acceptor ratio (1:2, 1.45:1, 3:1), Fe(II):Mn(II) ratio (1:9, 3:7, 5:5) demonstrated that the highest nitrate removal ratio was 100%, 49.6% (Mn(II)) and 100% (Fe(II)) under the conditions of electron donor:electron acceptor ratio of 3:1, Fe(II):Mn(II) ratio of 5:5. Mn(II) and Fe(II) as electron donor was tested for the effects on denitrification in the IBF reactor. Optimal conditions were obtained at an electron donor:electron acceptor ratio of 2:1, hydraulic retention time of 12 h and Fe(II):Mn(II) ratio of 5:5 with the highest removal ratio of nitrate-N (100%), Mn(II) (50.25%) and Fe(II) (99.2%). Results suggest that the optimal condition obtained from the IBF was feasible.