A novel water-soluble hexa-peri-hexabenzocoronene (HBC) derivative with peripheral functional groups, which facilitates a two-step assembly process in water that includes fiber formation via Pi stacking and subsequent peptide probing via electrostatic interactions, is reported. In the first step, the HBC derivative self-assembles into water-soluble red-fluorescent fibers that serve as templates for further functionalization with biomolecules. In the second assembly step, the peripheral functional groups bind green-fluorescent fluorescein-conjugated peptides, leading to the formation of well-defined fibers that were visualized as dual-color fibers in double-fluorescence imaging.