Solutions and powders formed from salmon sperm deoxyribonucleic acid (DNA) reacted with the cationic surfactant cetyltrimethylammonium chloride (CTMA-Cl) incorporated fluorescent rhodamine molecules: anionic sulforhodamine 640 (SRh) or cationic/zwitterionic rhodamine 640 perchlorate (RhP). The role of the cationic surfactant in the interaction between rhodamine dye and DNA-surfactant molecules has been investigated in both solution and solid state using optical spectroscopy and electrophoresis. Unexpectedly, the dye molecules did not interact directly with DNA, rather the DNA double helix acted as a template for the interaction between dye molecules and CTMA in the DNA/CTMA complex. The SRh and RhP molecules yield different fluorescence characteristics with increasing DNA/CTMA amount, indicating different configurations between the CTMA ligands.