In this study, glutaryl-7-aminocephalosporanic acid (ACA) acylase was immobilized on a silica gel modified with a mixture of 3-glycidoxypropyltrimethoxysilane (3-GPTMS) and NN-diisopropylethylamine (N,N-DIPEA). Unfortunately, the immobilized acylase showed very poor values for protein binding yield and activity. These results are believed to be caused by a discordance between the ring opening condition of epoxide groups and the immobilization condition for glutaryl-7-ACA acylase. Therefore, a new immobilization method for glutaryl-7-ACA acylase was introduced. Ethylenediamine, a strong alkaline reagent, was used as a spacer after epoxide silanization, and followed by glutaraldehyde as a crosslinking agent. The optimal conditions for each step of the immobilization procedure developed in the present study were as follows: epoxide silanization by a mixture of 30% (v/v) 3-GPTMS, 3% (v/v) N,N-DIPEA, and 10% (v/v) ethylenediamine and a 4-h reaction time between ethylenediamine and silica gel activated with epoxide silanization and crosslinking with 2% (v/v) glutaraldehyde. The immobilized glutaryl-7-ACA acylase was also tested for long-term stability. It was found that the activity was about 62% of the initial value after reuse of 20 times.