Hierarchical supramolecular nanostructures are architecturally robust and functionally tunable, and therefore are widely utilized as functional building blocks for optoelectronic and biological applications. Herein, we report a simple and effective fabrication strategy to construct hierarchical biomolecular nanolayers comprising cholesterol and cholesteryl hemisuccinate for automatic alignment of liquid crystals (LCs) by using the programmed interfacial self-assembly of biomolecular amphiphiles on the polar electrode surface. The spontaneous formation of ultrathin biomolecular nanolayer was successfully achieved by simple doping and in situ self-organization of a small amount of steroid-based amphiphiles in the confined LC cell. The surface analysis demonstrated that the amphiphilic biomolecules of cholesterol and cholesteryl hemisuccinate formed hydrophobic self-constructed nanolayers with a dense surface coverage on the surface of indium tin oxide (ITO) electrode via hydrogen bonding. These nanolayers, bearing hydrophobic steroid groups and alkyl chains on their surface, induced uniform vertical alignment of LCs via van der Waals forces. Notably, the biomolecular nanolayer containing cholesteryl hemisuccinate exhibited more stably oriented LCs than cholesterol-based nanolayer at the same concentration owing to the strong interfacial hydrogen bonding with polar ITO electrode surface. The electro-optical properties of the LC cells containing the steroid-based amphiphiles revealed that hierarchical self-constructed nanolayers based on the amphiphilic biomolecules afforded low threshold voltage and fast response time comparable to those of conventional polyimide alignment layer.