The precise alignment of liquid crystals (LCs) is crucial in the fabrication of LC devices because this arrangement can determine the performance of optoelectronic devices. Conventionally, LC alignment is achieved using a thin layer of elaborate polyimide materials. However, these materials require not only complicated synthetic processes using significant amounts of toxic chemicals, but also a time-consuming high-temperature curing process involving a long period of energy consumption. Thus, the development of environmentally sustainable alignment materials is a fundamental way to conserve energy and reduce the use of hazardous substances. Herein, we present an environmentally sustainable strategy to fabricate a functional vertical alignment layer for nematic LCs through interfacial self-assembly of chlorophyll biomolecules. A novel functional alignment layer was prepared using a simple and environmentally-friendly approach by doping chlorophyll extracted from plants, which are abundant in nature, into LC medium. It has been experimentally proven that amphiphilic chlorophyll biomolecules were self-assembled on the indium tin oxide surface through hydrogen bonding between a porphyrin ring and hydroxyl group, and therefore the stable homeotropic alignment of LC was achieved through the van der Waals interaction between the hydrocarbon tail and LC molecule. In addition, the nanoscale self-assembled alignment layer of chlorophyll molecules exhibited color-switchable behavior under visible and ultraviolet light. This simple and eco-friendly approach provided excellent electro-optical properties comparable to those of a commercial polyimide layer, while achieving a very stable and cost-effective vertical alignment layer capable of color switching.