In this study, the enantiomeric separation of antidepressant drug fluoxetine (FLX) was carried out using chiral selector-immobilized polyethersulfone (PES) membranes. For this purpose, the chiral selectors BSA, Candida rugosa lipase (CRL) and alpha-cyclodextrine (alpha-CD) were adsorbed on PES membranes by the static adsorption at different pH values. Chemical and electrokinetic characterizations of modified membranes were done by Fourier transforms infrared (FTIR) spectroscopy and by an electrokinetic analyzer (EKA), respectively. FTIR and EKA analysis were particularly employed in order to evaluate the immobilization efficiency. Racemic FLX was ultrafiltrated with the chiral selective membranes using the stirred ultra filtration cell. The effectiveness of chiral separation was characterized in terms of the values of enantiomeric excess (ee%) in permeate. During the ultra filtration, the samples taken from permeate were analyzed with liquid chromatography system using Chiral-pak AD-H column for the determination of R-FLX and S-FLX enantiomers. The ee(s) values of BSA and CRL in the permeate were approximately equal.