The formation of bilayers and the production of lipid-free vesicles from a naturally occurring, strongly amphiphilic protein, the hydrophobin HFBI was reported. To achieve protein bilayers with a hydrophobic core, a microfluidic cross-channel geometry made of hydrophobic material (poly(dimethyl)siloxane, PDMS) was utilized. The proteins contained in the fingers� buffer solution quickly cover the formed interfaces. Upon contact of the two fingers, a straight interface is formed spontaneously that increases with time until a constant bilayer area with contact angle is reached, which indicates a balance between the bilayer tension and the tensions of the individual. To further probe the nature of the hydrophobic bilayer core, the capacitance of the HFBI bilayer with as continuous medium is determined. Bilayers with a hydrophilic core were achieved using a hydrophilic material for the same microfluidic chip geometry and inverting continuous and finger phases. Since hydrophobins are natural occurring proteins also present in fungal spores, a better biocompatibility than for vesicles made out of block copolymers or nanoparticles is expected. Hydrophobin vesicles thereby serve as a first example demonstrating the proof-of-concept of a pure protein platform for synthetic biology or drug delivery.