Symmetrical and unsymmetrical cyanine dyes are used in different applications due to their different fluorogenic behaviors toward bio-macromolecules and micro-environments. In the present paper, computational studies on these dyes reveal that the potential energy of the electronic excited state is controlled by C-C bond rotational motion, which causes mainly nonradiative deactivation, according to the activation energies for the rotation. The rotations of different C-C bonds in the molecules have quite different rotational activation energies. Symmetrical dyes (Cy) possess an obviously higher rotating energy barrier as well as a larger energy gap compared to unsymmetrical dyes (TO). The C-C bond rotation close to the quinoline moiety of unsymmetrical thiazole orange (TO) allows the dye to possess the lowest energy barrier and also the lowest energy gap. This rotation plays a major role in reducing fluorescence quantum yields and providing a low fluorescent background in the free states of the unsymmetrical cyanine dyes. The results might provide a foundation for the interpretation of the behavior of the dyes and are useful for the future design of new cyanine fluorophores.