Dilute glutathione solutions containing NaCl need to be purified and concentrated. Nanofiltration (NF) would be a good solution, provided that it could completely reject glutathione while permeating NaCl entirely. Commercially available NF membranes are incapable of meeting both of these targets. Therefore, in this study, we modified a commercially available NF membrane by the layer-by-layer (LBL) assembly of alternating polyelectrolyte on thin films, to increase both the glutathione rejection and the membrane selectivity for NaCl over glutathione. Poly(styrene sulfonate) (PSS) was selected as the anionic polyelectrolyte, and poly(diallyldimethylammonium chloride) (PDADMAC) was chosen as the cationic polyelectrolyte. The separation performances of the modified membranes were studied with 1 g/L synthetic glutathione solutions containing 2 g/L NaCl at a pressure of 800 kPa. The results demonstrated that the glutathione rejection increased from 74% (unmodified) to 97.27% ([PSS/PDADMAC](10)/PSS) and the selectivity for NaCl over glutathione increased from 2.8 (unmodified) to 23 ([PSS/PDADMAC](10)/PSS).