Separation of l-glutamine (l-Gln) from Gln fermentation broth by nanofiltration (NF) was investigated with NTR7450 NF membrane. The effects of various experimental parameters such as transmembrane pressure, pH and concentration of broth on the rejection of l-Gln and l-glutamate (l-Glu) showed that NTR7450 was able to effectively separate l-Gln and l-Glu when the appropriate conditions were chosen. Moreover, the rejection of l-Gln and l-Glu in a single amino acid solution was also studied with the variation of pH, concentration, and physicochemical parameters. It was demonstrated that deviations in the rejection of fermentation broth from a single l-Gln or l-Glu solution were mainly caused by the complex ionic composition of the real fermentation broth.