The separation of a compound of interest from its structurally similar homologues is an important and challenging problem in producing high-purity natural products, such as the separation of genistein from other soybean isoflavone aglycone (SIA) homologues. The present work provided a novel method for separating genistein from its structurally similar homologues by ionic liquid (IL)-based liquid-liquid extraction using hydrophobic IL-water or hydrophilic IL/water-ethyl acetate biphasic systems. Factors that influence the distribution equilibrium of SIAs, including the structure and concentration of IL, pH value of the aqueous phase, and temperature, were investigated. Adequate distribution coefficients and selectivities over 7.0 were achieved with hydrophilic IL/water-ethyl acetate biphasic system. Through a laboratory-scale simulation of fractional extraction process containing four extraction stages and four scrubbing stages, genistein was separated from the SIA homologues with a purity of 95.3% and a recovery >90%.