Molecular self-assemblies offer a promising strategy for the synthesis of advanced materials for the construction of novel chiral sensing systems, and latest innovations on such self-assemblies are focused on simple building blocks in biology such as nucleic acids, lipids, and peptides. Herein, the self-assemblies of diphenylalanine (FF) and oxalic acid (OA) were prepared as the chiral sensing device for the recognition of tryptophan (Trp) isomers. Interestingly, the self-assemblies composed of OA and FF with different charging states (neutral, positively charged, and negatively charged) exhibited quite different morphologies, resulting in significantly different chiral recognition ability toward the Trp isomers. Also, in this work, the temperature sensitivity and chiral selectivity of the proposed FF-OA self-assemblies were also studied. From a practical point of view, the FF-OA self-assemblies were finally applied for the determination of precise levels of d-Trp in the racemic mixture of Trp isomers.