The sugars produced by enzymatic hydrolysis of rice straw are separated using cross-flow diafiltration in this study. The effects of membrane type, membrane pore size, cross-flow velocity and transmembrane pressure on the filtration flux, sugar rejection, and sugar mass flux transported to the filtrate are discussed. The filtration flux increases with increasing cross-flow velocity or transmembrane pressure. When the membrane made of mixed cellulose ester (MCE) is used, over 70% filtration resistances are caused by the membrane fouling; while the resistance due to virgin membrane is dominant when regenerated cellulose (RC) membranes are used. A force balance model is applied to relate the filtration flux and filtration resistance to operating conditions. The calculated data of filtration flux based on this model agree fairly well with experimental data. In addition, a theoretical model is used to explain the sugar transmission through the cake and membrane pores. The sugar rejection coefficient decreases with increasing cross-flow velocity because of the effect of cake reduction. This effect is more significant when the MCE membrane is used. Comparing the sugar mass flux transported into the filtrate, it is more effective for sugar purification by using 10 kD RC membrane and under higher transmembrane pressures.