With the improvement of wastewater discharge standards, wastewater treatment plants (WWTPs) are continually undergoing technological improvements to meet the evolving standards. In this study, a quartz sand deep bed denitrification filter (DBDF) was used to purify WWTP secondary effluent, utilizing high nitrate nitrogen concentrations and a low C/N ratio. Results show that more than 90% of nitrate nitrogen (NO3-N) and 75% of chemical oxygen demand (COD) could be removed by the 20th day of filtration. When the filter layer depth was set to 1,600 mm and the additional carbon source CH3OH was maintained at 30 mg L−1 COD (20 mg L−1 methanol), the total nitrogen (TN) and COD concentrations of DBDF effluent were stabilized below 5 and 30 mg L−1, respectively. Analysis of fluorescence revealed that DBDF had a stronger effect on the removal of dissolved organic matter (DOM), especially of aromatic protein-like substances. High throughput sequencing and qPCR results indicate a distinctly stratified microbial distribution for the main functional species in DBDF, with quartz sand providing a good environment for microbes. The phyla Proteobacteria, Bacteroidetes, and Chloroflexi were found to be the dominant species in DBDF.