Biomolecule-based materials have attracted increasing attention for the fabrication of environmental-friendly triboelectric nanogenerators (TENGs) owing to the inductive effect with strong electron-donating ability from NH₂ groups. However, understanding the relationship between molecular packing and performance is extremely challenging due to the complex structures, thus limiting the designability of highly efficient triboelectric biomaterials at the molecular level. Here, we describe minimalistic yet mechanically robust ordered amino acid crystals formed by physical vapor deposition and utilized as positive triboelectric materials. The morphologies and electrostatic potential could be optimized by rationally tuning the chirality-dependent supramolecular spatial arrangement, which results in a 50% increase in the output voltage of the corresponding TENGs. Interestingly, the electric field-induced directional molecular packing results in a surface comprising charged amino groups that could easily lose electrons for charge separation, which more than doubled the output voltage of TENGs. The amino acid crystal-based TENG showing enhanced performance could serve as a micro-power source to drive light-emitting diodes and as a sensor to detect body movements. Our work provides insight into exploring the triboelectric effect of biomaterial crystals and paves an avenue to high-performance electric devices for bioengineering applications by designing biomolecule-based functional materials.