The mass transfer of substrates through a bio-catalytic membrane layer is a key issue in determining the performance of beta-galactosidase-catalyzed conversion of lactose in a hollow fiber membrane reactor (HFMR) system. An investigation on the effect of solutes mass transfer through a bio-catalytic membrane layer was carried out using the coupled mass transfer-reaction model. Product formation was reduced at a trans-membrane pressure (TMP) of higher than 0.5 bar. Meanwhile, the concentration polarization modulus of solutes rapidly increased with higher TMP and this result suggests the formation of gel layer, which reduced bio-catalysis rate at higher applied TMP. The concentration profile of solutes or substrates on the bio-catalytic membrane surface, which determines the rate of reaction was reduced due to mass transfer limitation. This investigation highlights that the formation of substrate-beta-gal complex in an immobilized system is influenced by the mass transfer behavior of its substrate.