Glutaryl-7-aminocephalosporanic acid(GL-7-ACA) acylase is an important enzyme for the production of 7-ACA (7-aminocephalosporanic acid). For an efficient immobilization of GL-7-ACA acylase, various carriers were tested. A high-porous hydrophilic carrier (FPHA) among various carriers tested was found to be the best for the immobilization of GL-7-ACA acylase. In order to develop an effective immobilization method of GL-7-ACA acylase, the parameters that affect the immobilization of GL-7-ACA acylase were also investigated under different conditions of buffer solution and different concentrations of glutaraldehyde. The highest value of GL-7-ACA acylase activity (70 Unit/g-matrix) was obtained when immobilized with 1% glutaraldehyde in a 0.1M Tris buffer (pH 8.0). Also, in order to enhance the activity of the immobilized GL-7-ACA acylase, unreacted aldehyde groups were quenched by reaction with a low molecular weight agent such as L-lysine after immobilization. The highest activity of immobilized GL-7-ACA acylase was obtained at 0.1% of L-lysine. The immobilized GL-7-ACA acylase was tested for long-term stability and it was found that the activity was retained at about 62% of the initial value after 72 times of reuse at 25 ℃.