For practical application, the stability of permeabilized Ochrobactrum anthropi SY509 needs to be increased, as its half-life of enzymatic denitrification is only 90 days. As the cells become viable after permeabilization treatment, this can cause decreased activity in a long-term operation and induce breakage of the immobilization matrix. However, the organic solvent concentration causing zero cell viability was 50%, which is too high for industrial application. Thus, whole-cell immobilization using glutaraldehyde was performed, and 0.1% (v/v) glutaraldehyde was determined as the optimum concentration to maintain activity and increase the half-life. It was also found that 0.1% (v/v) glutaraldehyde reacted with 41.9% of the total amine residues on the surface of the cells during the treatment. As a result, the half-life of the permeabilized cells was increased from 90 to 210 days by glutaraldehyde treatment after permeabilization, and no cell viability was detected.