The short-cut nitrification and denitrification process of a polyurethane biofilm reactor was studied at four different carbon to nitrogen ratios (C/N ratios) (C/N =15:1, 10:1, 5:1 and 1.8:1) with focus on reactor performance and microbial community composition. Results showed that when the influent carbon/nitrogen ratio decreased from 15 to 10, 5 and 1.8, the nitrite accumulation rate was increased from 56.1% to 62.3%, 72.3% and 83.2% after 100 d operation. System achieved the optimal removal efficiency at an influent carbon/nitrogen ratio of 10 with 99.1% of ammonia removal and 91.0% of total nitrogen removal. Simultaneous nitrification and denitrification occurred in the polyurethane biofilm system at different influent carbon/nitrogen ratio. As the influent carbon/nitrogen ratio decreased from 15 to 10, 5 and 1.8, the efficiency of simultaneous nitrification and denitrification was gradually reduced. The microbial biofilm analysis indicated that the microbial diversity of biofilm with an influent carbon/nitrogen ratio of 15 was significantly higher than that in other conditions. The predominant ammonia oxidizing bacteria and nitrite oxidizing bacteria were Nitrosospira sp. and Nitrospira sp., respectively, and the predominant denitrifying bacteria were Pseudomonas sp.