A new microorganism immobilization method involving preparation and gelation of waterborne polyurethane (WPU) was developed. The WPU prepolymer was synthesized by polyether polyol, isophoronediisocyanate (IPDI), 2-hydroxylethyl methacrylate (HEMA) were prepared by radical polymerization. The feasibility of the proposed immobilization method was tested by nitrifying bacteria. The effects of WPU prepolymer and gelation process on the activity of nitrifying bacteria were investigated by relative respiratory activity. The microbial growth and distribution of immobilized nitrobacteria were observed with a scanning electron microscope (SEM) before and after acclimation. The results verified that the waterborne polyurethane is an ideal microbial immobilization support material and has a great potential for a variety of biological wastewater treatment applications.