Biofuel, that is, n-butanol, production from plant-based biomass represents a renewable energy resource that can be a substitute for fossil fuel. Thus, it is important to study the separation of n-butanol from its aqueous solution to obtain a high-purity product. Liquid-liquid extraction is a beneficial process in alcohol production to recover alcohol from the aqueous medium. Liquid-liquid equilibrium (LLE) data are needed for rational design and optimal separation operation, such as biological separation and extraction processes. The objective of this study is to determine LLE data for quaternary systems of n-butanol, methyl isobutyl ketone (MIBK), water, and a biological buffer at 303.15 K and atmospheric pressure (P = 0.1 MPa). The effect of biological buffer addition into aqueous solution was also studied. Three different biological buffers, 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid (EPPS), 4-morpholinepropanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), were applied in this study for the comparison. The separation factor and distribution coefficient were evaluated in this study to interpret the extraction performance of n-butanol. According to the experimental results, the extraction performance was in the order of HEPES > EPPS > MOPS, describing the strength of buffering-out as well. Furthermore, it was found that the aid of a biological buffer in the LLE systems consisting of n-butanol, MIBK, and water resulted in better extraction performance than that of the LLE systems in the absence of a biological buffer. The reliability of the experimental LLE tie-line data was satisfactorily ascertained by the Hand correlation. The non-random two-liquid and universal quasi-chemical thermodynamics models correlated well the experimental LLE tie-line data with the root mean square deviation of less than 1%. The binary interaction parameters of both models were also obtained in this study.