Discharge of wastewater with high organic materials demands a significant development of polymeric ultrafiltration membrane for efficient treatment. Among the various nanomaterials used as additives in the polymeric membrane, copper oxide nanoparticles (CuO NPs) were particularly notable. The nanoparticles are available at an inexpensive cost, with vast surface area and ease of surface functionalization. However, the nanoparticle tends to agglomerate in the polymeric membrane due to the high surface activity of particles with smaller sizes. Surface modification of CuO NPs using amino acid L-cysteine as a stabilizer can overcome this problem. In this work, the surface modification process undergoes ultrasonication to create covalent anchoring at different ratios of L-cysteine to CuO NPs. Preparation of dope solution involves the addition of 18.0 wt.% polysulfone (PSf) and 5 wt.% polyvinylpyrrolidone (PVP). Then, the fabrication of the flat sheet membranes employs an immersion precipitation process. The TEM and FTIR analysis confirmed the presence of modified CuO NPs. Investigation using SEM, AFM, porosity, tensile strength, and zeta potential thoroughly characterized the membrane. Measurements of contact angle and water content uptake determined the membrane hydrophilicity. Evaluation of pure water flux (PWF), protein rejection, antifouling capability, and filtration performance of synthetic poultry wastewater propose the membrane separation performance. Data revealed that modified nanoparticles improved the ability of the membrane to resist fouling, and BSA rejection was above 95%. Accordingly, the PSf membrane with a 1:1 ratio of L-cysteine to CuO NPs showed the best UF performance.