The fluorescence resonance energy transfer (FRET) in a lipid bilayer system containing two different donors and one common acceptor at below and above transition temperature has been studied and all the FRET parameters are analyzed using steady state and time-resolved fluorescence spectroscopy. Using dynamic light scattering measurement, we have followed the process of preparation of small unilamellar vesicles, and by following the FRET parameters of C-153–Rh6G and C-151–Rh6G pairs inside SUVs at 16 °C and 33 °C (T_m = 23.9 °C) we have noticed that there is greater effect of temperature on the FRET parameters in case of the C-153–Rh6G pair than that of the C-151–Rh6G pair. Finally we have concluded that this difference is due to their different location inside the lipid bilayer in which fluidity of the long alkyl chain markedly affects the FRET parameters for C-153–Rh6G pair embedded inside a small unilamellar vesicle of size 20–50 nm.