In the past quarter century, applications of fluorescence resonance energy transfer (FRET) have grown dramatically, and the technology has become an indispensable tool in a wide variety of biological and biophysical domains. It is utilized, in order to acquire information on the conformational changes that occur in single molecules. By utilizing the fluorescence correlation spectroscopy, the pharmaceutical sector has also built huge fluorescence detection systems with extremely small sample sizes, reaching down to the level of single molecules. The fluorescence resonance energy transfer (FRET) between the two dyes, Acriflavine (Acf) and Rhodamine B (RhB), were examined in solution. Energy transfer was observed in fluorescence resonance imaging solutions containing Acriflavine and Rhodamine B with different concentrations of the acceptor RhB dye in the range of (1.5×10-5 M to 3.5×10-5M). Studies using Both UV–Vis absorption and fluorescence spectroscopy demonstrated that the two dyes, when dissolved in solution, appear largely as monomers.