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The design and scalable construction of robust ultrathin protein membranes with tunable separation properties remain a key challenge in chemistry and materials science. Here, we report a macroscopic ultrathin protein membrane with the potential for scaled-up fabrication and excellent separation efficiency. This membrane, which is formed by fast amyloid-like lysozyme aggregation at air/water interface, has a controllable thickness that can be tuned to 30–250 nm and pores with a mean size that can be tailored from 1.8 to 3.2 nm by the protein concentration. This membrane can retain > 3 nm molecules and particles while permitting the transport of small molecules at a rate that is 1~4 orders of magnitude faster than the rate of existing materials. This membrane further exhibits excellent hemodialysis performance, especially for the removal of middle-molecular-weight uremic toxins, which is 5~6 times higher in the clearance per unit area than the typical literature values reported to date.