Glucose and enzyme in a high concentration enzymatic hydrolysis suspension were separated using hollow fiber cross-flow diafiltration. The operating condition effects on the filtration performance are discussed. Over 86% of the enzymes were retained using a 10 kDa polysulfone membrane. Most glucose molecules penetrated through the membrane into the filtrate. The major filtration resistances are due to the cake formation on the membrane surface and enzyme blocking in the membrane pores in addition to the appreciable virgin membrane resistance. The cake mass is correlated with the drag force ratio tangential to the filtration directions and the average specific cake filtration resistance is expressed as a power-function of the transmembrane pressure with a cake compressibility of 0.3. The empirical equation related the resistances due to internal membrane fouling to the operating conditions established by conducting experimental data regression. The filtration flux can be estimated accurately by substituting the results calculated using the empirical equations into the basic filtration equation. An increase in cross-flow velocity or transmembrane pressure leads to higher filtration flux. The filtration flux increases ca 2-fold as the cross-flow velocity increases from 0.3 to 1.5 m/s under a transmembrane pressure of 60 kPa. The filtration flux increases over 3-fold as the transmembrane pressure increases from 20 to 100 kPa. Two kinds of modified operating methods are used to improve the filtration flux. A pulse feeding method may increase the filtration flux by 25%, while the step-increase pressure method improves the filtration flux by over 34%. Forming a thin cake under low pressure in the early filtration period has great potential to significantly mitigate membrane pore blocking and enhance filtration flux.