Developing substrate with carbon release properties is helpful to enhance nitrogen removal in low C/N ratio wastewater treatment. In this study, substrates with and without adding carbon source and microorganism were prepared to treat the drainage effluent from a wastewater treatment plant (WWTP), the difference in nitrogen removal were investigated. The results showed that adding a carbon source and microorganism to substrates could not only increase the amount of chemical oxygen demand (COD) released, but also enhance the adsorption capacities of NH4+-N and NO3–-N. The carbon release process followed the first-order kinetic equation. A nitrogen removal model consisting of four phases of diffusion, adsorption, assimilation and transformation was proposed. In the short term, nitrogen was mainly removed by adsorption; adding microorganism contributed to enhance nitrification and denitrification. In the long-term, nitrogen removal performances were similar whether microorganism was added or not, and microbial species on the surface of substrates were similar. This work suggested when using substrate to treat wastewater for nitrogen removal, preparing a substrate with excellent property for biofilm formation was the most important factor.