Bovine serum albumin(BSA) as a chiral ligand was captured uniformly throughout a porous hollow-fiber membrane at a level of 250mg/g by the polymer chains grafted onto the membrane. Subsequently, crosslinking of BSA with a 0.05%(w/w) of glutaraldehyde in a Tris-HCl buffer (pH 8) for 5 h was effective in stabilizing the amount of BSA immobilized at a level of 200 mg/g. A solution of DL-Tryptophan in Tris-HCl buffer asa mobile phase permeated the crosslinked-BSA multilayered membrane. Therefore on experimentation is BSA immobilized hollow fiber is applicable for the chiral separation of enantiomers in a mobile phase with various pH values and organic modifiers.