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Bioretention has been widely used in urban non-point source (NPS) pollution management for effectively reducing downstream pollution loads and peak flows. However, nitrogen (N) removal in conventional bioretention systems has been uniformly unstable and highly variable due to a lack of anaerobic denitrification. To improve the stability and effectiveness of N removal, two bioretention columns with bilayer media (C1 and C2) were designed. High permeability quartz sand (~2 mm diameter) was used as the upper media, and low permeability modified media (~0.6 mm diameter, adding 5% organic substance) as the lower media. The bilayer media structure formed an anaerobic zone for promoting denitrification processes. The results showed that the retrofitted columns performed well and that the removal efficiencies of various forms of N were considerably enhanced to 76.8%-95.3%, 85.1%-98.3%, and 87.5%-97.4% for TN, NH 4 + -N, respectively. Additionally, copying numbers of the denitrification functional genes detected via FQ-PCR in the lower media of C1 and C2 were accounted for 46.06% and 44.16% of the 16S rDNA gene, respectively. These results confirmed the presence of anaerobic denitrification processes. Consequently, bilayer media bioretention systems are worth promoting in cities where nitrogen in urban runoff poses a threat to the receiving surface water, due to the systems' remarkable performance in nitrogen removal, simple structure, and easy implementation.3 - -N, respectively. Additionally, copying numbers of the denitrification functional genes detected via FQ-PCR in the lower media of C1 and C2 were accounted for 46.06% and 44.16% of the 16S rDNA gene, respectively. These results confirmed the presence of anaerobic denitrification processes. Consequently, bilayer media bioretention systems are worth promoting in cities where nitrogen in urban runoff poses a threat to the receiving surface water, due to the systems' remarkable performance in nitrogen removal, simple structure, and easy implementation., (Copyright © 2019 Elsevier B.V. All rights reserved.)