Functionalized microfiltration (MF) membranes are becoming increasingly popular in biotechnology and pharmaceutical industries. Affinity based separation using functionalized MF membranes are emerging as a potential alternative to conventional bead-based column chromatography due to their hydrodynamic advantages. Functionalization of MF membranes offer choices of attaching the active groups or moieties on different types of materials (hydrophilic, hydrophobic), environments (external surface, internal pore surface) and dimensions (pore size varying from 0.05 to 10 ƒÝm).This study deals with functionalization of microfiltration (MF) membranes within the pore structures and their applications in the field of bio-separations. Wide pore structure of MF membranes allows easy access of the active groups/moieties to the target molecules, under convective flow condition. Therefore, the mass transfer resistance, and hence the processing time per unit volume of feed is less. This study describes functionalization of MF membranes with active groups. Once the pore surface of the membrane is activated, further attachments of groups, moieties or molecules (ligands) are carried out. In this research work, avidin is attached as a ligand. Then, biotin-tagged protein is separated from mixture, purified and isolated from the matrix. The quality of the isolated pure protein is analyzed and compared with that obtained by conventional bead-based column chromatographic separation. Characterizations of the functionalized membranes are also presented in this study. Characteristic fouling of the membrane matrix associated with bio-separations is also analyzed critically. Some, simplified mathematical model is presented to describe the transport of bio-molecules within the membranes.