Surfaces that resist nonspecific protein adsorption in a complex biological milieu are required for a variety of applications. However, few strategies can achieve a robust antifouling coating on a surface in an easy and reliable way, regardless of material type, morphology, and shape. Herein, the preparation of an antifouling coating by one‐step aqueous supramolecular assembly of bovine serum albumin (BSA) is reported. Based on fast amyloid‐like protein aggregation through the rapid reduction of the intramolecular disulfide bonds of BSA by tris(2‐carboxyethyl)phosphine, a dense proteinaceous nanofilm with controllable thickness (≈130 nm) can be covered on virtually arbitrary material surfaces in tens of minutes by a simple dipping or spraying. The nanofilm shows strong stability and adhesion with the underlying substrate, exhibiting excellent resistance to the nonspecific adsorption of a broad‐spectrum of contaminants including proteins, serum, cell lysate, cells, and microbes, etc. In vitro and in vivo experiments show that the nanofilm can prevent the adhesion of microorganisms and the formation of biofilm. Compared with native BSA, the proteinaceous nanofilm coating exposes a variety of functional groups on the surface, which have more‐stable adhesion with the surface and can maintain the antifouling in harsh conditions including under ultrasound, surfactants, organic solvents, and enzymatic digestion.