The flux decline and rejection behavior in cross-flow ultrafiltration (UF) of BSA/lysozyme (Ly) mixtures, with and without ultrasound, were investigated in upward and downward modes. Polysulfone (PS) and polyvinylidine fluoride (PVDF) ultrafilters were selected. Experiments were conducted at different pH values (4.9–11), applied pressures (2–4 atm), added NaCl concentrations (0.01–1 g/L), and ultrasonic powers (180–250 W). It was shown that PS membrane yielded higher flux and lower Ly rejection than PVDF membrane. Low rejection of Ly was achieved at pH near isoelectric point of Ly (11.0), particularly under the conditions of lower applied pressures and ionic strengths. The flux and Ly rejection were strongly affected by the solution environment. BSA was almost retained in the retentate, but its existence had a significant effect on permeate flux. The flux was enhanced with ultrasound and the enhancement increased with increasing ultrasonic power at 25 kHz, likely due to the change of molecular sizes of the proteins under ultrasonic irradiation. With ultrasound, the level of flux enhancement in the upward mode was slightly better than that in the downward mode.