Effectiveness of Bovine Serum Albumin (BSA) as chiral recognizing protein in enantiomers separation by ultrafiltration technique was studied by immobilizing BSA on the membrane and incorporating BSA as semi-interpenetrating network in the membrane matrix. Separation of racemic tryptophan solution was performed in closed loop cross flow ultrafiltration using BSA immobilized polysulfone membrane and polysulfone membrane having BSA semi-IPN network. The volumetric flux (Jv), the solute flux (Js), the separation factor (), and the enantiomeric excess (%ee) of two types of membranes at different trans-membrane pressures and permeation times were determined. BSA semi-IPN membrane exhibits higher volumetric as well as solute fluxes compared to BSA immobilized membrane. Separation factor () to the order of 1.89 was achieved with BSA immobilized membrane after 8h of ultrafiltration and in the same duration BSA-IPN membrane exhibited separation factor () to the order of 1.62. BSA immobilized membrane exhibits higher enantiomeric excess (30.8%) compared to BSA semi-IPN membrane (23.8%) after 8hrs. BSA molecules available on membrane as immobilized or as semi-IPN under go complexion with tryptophan enantiomers differently. BSA immobilized membrane performed better separation and enantiomeric purity; however, the solute flux of the membrane decreases.