The interaction of dyes with a sulfonated Gemini surfactant was investigated in aqueous solution using Förster resonance energy transfer with acridine orange (AO) as a donor and rhodamine B (RhB) as an acceptor. Surface tension results showed that AO and RhB have different effects on the self-assembly of the Gemini surfactant, with AO giving a higher critical micelle concentration (cmc) and lower surface tension, while the opposite was observed for RhB. Energy transfer from AO to RhB was observed in the presence of the surfactant, and the energy transfer efficiency initially improved with increased surfactant concentration but then decreased significantly when the surfactant reached a higher concentration due to the formation of larger aggregates, which increased the average distance between AO and RhB. Dynamic light scattering demonstrated the existence of these large aggregates. Moreover, simulations using dissipative particle dynamics supported the experimental results.