The invention discloses a Fe<0>-containing high-polymer biological membrane filling as well as a preparation method and application thereof. The filling mainly comprises a composite biological membrane, a multi-tooth net-shaped sphere matrix and Fe<0>-containing high-polymer gel, wherein the composite biological membrane rich in nitration and nitrosation bacteria covers the surface of the multi-tooth net-shaped sphere matrix, the multi-tooth net-shaped sphere matrix is filled with the Fe<0>-containing high-polymer gel, and the Fe<0>-containing high-polymer gel is capable of efficiently adsorbing PO4<3> and NO3 and simultaneously releasing Fe<2+> ions so as to promote the microbial activity in the biological membrane. The surface of the sphere matrix is subjected to multi-tooth embossing treatment, so that the biofilm formation of a filling matrix is easily realized; and a sphere can be detached, the high-polymer gel filled in the sphere can be conveniently taken out for regeneration, and the adsorption capacity of the regenerated gel is not obviously decreased. The biological membrane filling has the capacity of simultaneously removing multiple pollutants such as COD, NH<4+>, NO3, TN and TP, is particularly applicable to the treatment of domestic wastewater, and has wide application prospects.