In this work, TiO2, which was modified by glutaraldehyde, was adopted as the carrier; the penicillin G acylase (PGA) was immobilized and the influence of immobilized conditions, such as pH of solution, the concentration of PGA, the immobilization temperature, and the reaction time, on the catalytic performance of the immobilized PGA was investigated and optimized. During this process, potassium penicillin G (PG) was chosen as substrate, and the quantity of 6‐aminopenicillanic acid (6‐APA) produced by PG at the temperature of 25 °C for 3 Min in neutral solution was conscripted as the evaluation foundation, indexes, containing the loading capacity (ELC), the activity (EA), and activity retention rate (EAR), were calculated based on quantities of produced 6‐APA and compared with finding out the suitable conditions. Results showed that when the solution pH, PGA concentration, immobilization temperature, and reaction time were 8.0, 2.5% (v/v), 35 °C, and 24 H, respectively, ELC, EA, and EAR presented optimal values of 9,190 U, 14,969 U/g, and 88.5% relatedly. After that, the stability and reusability of immobilized PGA were studied, and the results documented that the pH resistance, thermal stability, and storage stability of immobilized PGA were significantly improved. This work provided technique support for the practical application of immobilized PGA carrier.