Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase is an enzyme that converts GL-7-ACA to 7-aminocephalosporanic acid, a starting material for semisynthetic cephalosporin antibiotics. In this study, optimal conditions for the immobilization of GL-7-ACA acylase were determined by experimental observations and statistical methods. The optimal conditions were as follows: 1.1 M phosphate buffer (pH 8.3) as buffer solution, immobilization temperature of 20 degrees C, and immobilization time of 120 min. Unreacted aldehyde groups were quenched by reaction with a low-molecular-weight material such as L-lysine, glycine, and ethanolamine after immobilization in order to enhance the activity of immobilized GL-7-ACA acylase. The activities of immobilized GL-7-ACA acylase obtained by using the low-molecular-weight materials were higher than those obtained by immobilized GL-7-ACA acylase not treated with low-molecular-weight materials. In particular, the highest activity of immobilized GL-7-ACA acylase was obtained using 0.4% (v/v) ethanolamine. We also investigated the effect of sodium cyanoborohydride in order to increase the stability of the linkage between the enzyme and the support. The effect on operational stability was obvious: the activity of immobilized GL-7-ACA acylase treated with 4% (w/w) sodium cyanoborohydride remained almost 100% after 20 times of reuse.