Toluidine blue (TB) dye molecules are intensively utilized for large-area photophysics applications such as carcinoma detection, photoinactivation of bacteria, biosensors, and photovoltaic cells. Understanding the nature of the TB aggregation state becomes an essential point of the research process in order to know the structure-function relationship and to foresee technological applications of this class of metachromatic-dye molecules. However, no structural information on toluidine blue is available in the literature, maybe because of the poor crystalline character of the aggregate. Here, we present the first structural determination of TB organic molecules using the energy dispersive X-ray diffraction technique. The investigation highlights dimeric arrangements of stacked molecules in antiparallel fashion, forming a superstructure of two dimers in a transverse arrangement. The behavior of the TB higher aggregates indicates that these dye molecules, in spite of repulsion due to similar charge (cationic dyes), undergo self-aggregation to form helical conformations.