Nanofiber membranes for chiral separation were prepared from chitin, which is the most abundant natural amino polysaccharide. The membrane showed chiral separation ability by adopting concentration gradient as a driving force for membrane transport. In other words, the chitin nanofiber membrane selectively transported the D-isomer of glutamic acid (Glu), phenylalanine (Phe), and lysine (Lys) from the corresponding racemic amino acid mixtures.