The process of simultaneous nitrification and denitrification (SND) of immobilized microorganisms in polyurethane form is discussed. The effect of different positions within the polyurethane carrier on microbial community response for the SND process is investigated by a combination of denaturing gradient gel electrophoresis profiles of the 16S rRNA gene V3 region and scanning electron microscopy. Results show that polyurethane, which consists of a unique porous structure, is an ideal platform for biofilm stratification of aerobe, anaerobe and facultative microorganisms in regard to the SND process. The community structure diversity response to different positions was distinct. The distributions of various functional microbes, detected from the surface aerobic stratification to the interior anaerobic stratification of polyurethane, were mainly nitrifying and denitrifying bacteria. Meanwhile aerobic denitrifying bacteria such as Paracoccus sp., Agrobacterium rubi and Ochrobactrum sp. were also adhered to the interior and surface of polyurethane. The SND process occurring on polyurethane foam was carried out by two independent processes: nitrogen removal and aerobic denitrification.