Nitrates in secondary effluent need further removal to ensure water quality and protect the aquatic ecosystem. A lab-scale continuous up-flow immobilized biofilter (I–BF) (15L) based on polyurethane foams (PUFs) carriers immobilized bacterial consortia was constructed for treating nitrate micro-polluted water (14-18mgN/L). The results showed that a low effluent total nitrogen concentration of 0.68 ± 0.11 mg/L and a high denitrification rate of 198.40 ± 4.50g/(m3·d) were obtained under a short hydraulic residence time (HRT) (1.8h). Shortening HRT decreased I–BF operating performance with a slight extent but enlarged the vertical spatial discrepancy in denitrification ability significantly. The high-throughput sequencing analysis revealed that Acidovorax belonging to β-proteobacteria was the dominant genus involving in denitrification in this sodium acetate-fed immobilized biofilter. The spatial community structure has changed as HRT decreased. The different microbial composition along the height of I–BF at short HRT could respond to vertical differentiation in denitrification performance, and meanwhile enable the strong resistance ability of I–BF to high load impact. The research on both the I–BF operating performance and the dynamic characteristics of the microbial community in the vertical spaces of the biofilter is likely to provide a foundation for the biofilter denitrification process in practice.