The highly complex microbial communities in biofilm play crucial roles in the pollutant removal performance of wastewater treatment plants (WWTPs). In the present study, using multi-omics analysis, we studied microbial structure, key enzymes, functional traits, and key metabolic pathways of pre-denitrification biofilter in an urban WWTP in China. The analysis results of metagenomic and metaproteomic showed that Betaproteobacteria and Flavobacteriia were dominant in biofilms. The integrated metagenomic and metaproteomic data showed that the expression of nitrogen metabolism genes was high, and the high proportion of denitrification module indicating that denitrification was the main nitrogen removal pathway. The most abundant denitrifying bacterial genera were: Dechloromonas, Acidovorax, Bosea, Polaromonas, and Chryseobacterium. And microorganisms with denitrification potential may not be able to denitrify in the actual operation of the filter. The integrated analysis of metaproteomic and metabolomic showed that there was a correlation between biofilm microorganisms and metabolites. Metabolomic analysis indicated that metabolic profiles of biofilms varied with layer height. This study provides the first detailed microbial communities and metabolic profiles in a full-scale pre-denitrification biofilter and clarifies the mechanism of denitrification.