In this work we have reported the formulation of a novel ionic liquid-in-oil (IL/O) microemulsion where the polar core of the ionic liquid, 1-ethyl-3-methylimidazolium n-butylsulfate ([C(2)mim][C4SO4]), is stabilized by a mixture of two nontoxic nonionic surfactants, polyoxyethylene sorbitan monooleate (Tween-80) and sorbitan laurate (Span-20), in a biological oil phase of isopropyl myristate (IPM). The formation of the microemulsion droplets has been confirmed from the dynamic light scattering (DLS) and phase behavior study. To assess the dynamic heterogeneity of this tween-based IL/O microemulsion, we have performed an excitation wavelength dependent fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to rhodamine 6G (R6G). The multiple donor-acceptor (D-A) distances, similar to 15, 30, and 45 angstrom, obtained from the rise times of the acceptor emission in the presence of a donor can be rationalized from the varying distribution of the donor, C480, in the different regions of the microemulsion system. With increasing the excitation wavelength from 375 to 408 nm, the contribution of the rise component of similar to 240 ps which results the D-A distance of similar to 30 angstrom increases significantly due to the enhanced contribution of the C480 probe molecules closer to the acceptor in the ionic liquid pool of the microemulsion.