Continuous enzymatic reaction has been proven as an efficient technique for several industrial applications. In this study, a type of hollow fiber membrane bioreactor where penicillin acylase entrapped within membrane pores was applied to continuously hydrolyze Penicillin G The influences of various operating conditions on immobilization and enzymatic reaction processes were assessed. A mathematical model of the reactor behaviour at steady state condition was also developed. The immobilization results show that penicillin acylase was entrapped more than 90% (100,000 u.a m(-2)). Due to the much smaller size of 6-APA compared to the membrane pore, the solute diffuses freely through the membrane. However, the immobilized enzyme membrane retained around 35% of the solute. In addition, K-m, of immobilized penicillin acylase (8.04 mM) was slightly higher than that of free penicillin acylase (7.75 mM). The theoretical results indicated that convective transport was the main mechanism of mass transport even in the case where flux was very low. Low flux rate is important to avoid gel formation or enzyme release from membrane pores and to maximize the degree of conversion.