The optical resolution of racemic mixtures of alpha-amino acids was performed using novel enantioselective polymer membrane to obtain optically pure isomers. The novel membranes were prepared by polymerizing a mixture of D-arginine, piperazine, and trimesoyl chloride in-situ interfacial on polysulfone ultrafiltration membrane. ATR-FTIR spectroscopy, Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), and Transmission Electron Microscopy (TEM) techniques were used to characterize the membranes and co-relate membrane morphology with membranes performance. The optical resolution performance of the membrane process was quantified by performing optical resolution of racemic arginine and asparagine in pressure driven separation mode at low pressures. The influence of monomers concentrations and their ratio in the polymerizing mixture on the flux and enantioseparation was studied. The observations indicated >91 percent enantiomeric excess (>91% ee) of L-arginine and >56 percent enantiomeric excess (>56% ee) of L-asparagine in the permeate of the racemic feed. The membrane prepared using equiconcentrated (2%) solutions of D-arginine, piperazine, and trimesoyl chloride exhibited highest enantiomeric excess (91%). At 91% ee the separation factor (alpha) for L-arginine was approximately 16.