Magnetic hydroxyl microspheres were synthesized and activated by using epoxyl chloropropane by means of suspension polymerization. The effects of amount of crosslink agent on the immobilization yield and activity yield of immobilized enzyme were investigated. The results showed that the magnetic polymer with crosslink density 30% could be used as an optimized immobilized penicillin G acylase (PGA) support. The apparent Michaelis constant Km and Vmax of the immobilized enzyme were 8.312×10−5 mol/l and 21.64 mol/min, respectively. The protecting immobilization was achieved by employing the interaction between the phenyl acetic acid (PAA) and PGA. It was found that activity yield of immobilized PGA was 1.0834-fold of the randomly immobilized enzyme. The optimal pH and temperature of the immobilized enzyme were 8.5 and 45◦C, respectively, for hydrolysis of penicillin G. Being applied for 80 times, the remained activity of the immobilized enzyme was about 94.2%.