Inspired by the bacterial biofilms and chorions of living organisms which are made by proteinaceous assemblies and functional for multi-applications, various artificial protein fibrils-based nanoporous films are developed, and show their potential applications in multiple fields. Here, a simple and environmental friendly method was identified to produce bovine serum albumin (BSA) nanofibrils based biofilms, through a combination of protein fibrillation and reverse dialysis. BSA nanofibrils formed biofilms through intermolecular interactions, the resultant biofilms showed tunable thickness by altering the initial protein amount, good stability in organic and salty solvents, transparency and fluorescence properties, hold high capacity of trapping different substances (e.g. nanomaterials, organic dyes, heavy-metal ions and enzymes), and further enabled applications in biologic water treatment and enzyme stabilization. Taken o-phenylenediamine as substrate, the trapped horseradish peroxidase showed a catalytic activity 9–38 folds higher than free ones in organic phase, together with enhanced stability. These protein nanofibrils-based films offered an attractive biologic platform to hybridize diverse materials for on-demand functions and applications.