Due to the complexity of fermentation mixtures, separation of solutes and secondary substrates has a special importance. Membrane processes such as nanofiltration may offer good alternatives for the separation of fermentation products or recovery of substrates. For an efficient separation, the membrane type and operating parameters such as feed flow rate, operating pressure, and feed should be optimized. In this study the separation and recovery of sugars were targeted. It was found that with an increase of the feed flow rate, permeate flux increased since the effect of concentration polarization was minimized. As a result, experiments were carried out at the highest possible flow rate. The effect of pressure was studied at five pressures (10-50 bar). It was found that there is a linear relationship between the pressure and permeate flux up to 30 bars. Beyond 30 bars the effect of pressure became less significant. Thus, 30 bars was chosen as the operating pressure. To investigate the effect of concentration, 1 to 10 weight percentage sucrose and glucose solutions were utilized. It was observed that with an increase in concentration, permeate flux decreased and rejections increased, finally reaching a limiting value. Binary solutions of sucrose and glucose were also studied. It was seen that the separation factor slightly decreased, probably due to a glucose-sucrose interaction. Experimental data were used with a mathematical model to predict the permeate flux and rejection. Good agreement of the predicted results was obtained with the experimental data for a 500 MWCO membrane.