An integrated fixed-biofilm activated sludge (IFAS) system was upgraded into a separated two-sludge denitrifying phosphorus removal system to treat domestic sewage with a low carbon/nitrogen ratio (C/N = 3.6). The system was operated in IFAS mode for 135 days and then converted to the separated two-sludge mode to perform denitrifying phosphorus removal for 110 days. With denitrification and phosphorus removal conducted using “single carbon source”, the experimental results show that the total inorganic nitrogen (TIN) and phosphorus (P) removal efficiencies were improved by 13.4% and 36.5%, respectively. As the activated sludge unit and biofilm unit were separated, Dechloromonas (4.10%) and Candidatus Accumulibacter (1.56%) were enriched in activated sludge system, while the nitrifiers Nitrospira and Nitrosomonas occupied the biofilm with proportions of 19.6% and 5.68%, respectively. The separated two-sludge denitrifying phosphorus removal system was characterized by higher nutrient removal and functional bacteria enrichment.