Membrane materials that resist nonspecific or specific adsorption are urgently required in widespread applications. In water purification, inevitable membrane fouling not only limits separation performance, but also remarkably increases operation requirements, and augments extra maintenance costs and higher energy consumption. In this work, we report a freestanding interfacial polymerization (IP) fabrication strategy for in-situ creation of asymmetric block copolymer (BCP) nanofilms with antifouling properties, greatly outperforming the conventional surface post-modification approaches. The resultant asymmetric BCP nanofilms with highly-dense, highly-hydrophilic polyethylene glycol (PEG) brushes, can be readily formed via a typical IP process of a double-hydrophilic BCP composed of an antifouling PEG block and a membrane-forming multiamine block. The asymmetric BCP nanofilms have been applied for efficient and sustainable natural water purification, demonstrating extraordinary antifouling capabilities accompanied with superior separation performance far beyond commercial polyamide nanofiltration membranes. The antifouling behaviors of BCP nanofilms derived from the combined effect of the hydration layer, electrostatic repulsion and steric hindrance were further elucidated by water flux and fouling resistance in combination with all-atom molecular dynamics simulation. This work opens up a new avenue for large-scale and low-cost creation of broad-spectrum, asymmetric membrane materials with diverse functional “defect-free” surfaces in real-world applications.