Enantioselective extraction of esmolol (ES) enantiomers was performed with tartaric acid derivatives and boric acid (BA) as chiral extractant. The single-stage extraction experiments were carried out to construct the extraction system for ES enantiomers through investigating type of tartaric acid derivatives, type of the organic solvent and temperature. The optimal conditions for extraction system were achieved, which involves 1,2-dichloroethane as organic solvent, iso-butyl-D-tartrate (DT) as chiral selector and 278 K for extraction temperature. A single-stage extraction model was established based on an interfacial reaction mechanism and proved to be reliable by verified experiments. Based on the single-stage extraction model and the law of mass conservation, a fractional extraction model was explored to simulate and optimize the fractional extraction process. The optimal operation conditions at temperature of 278 K were obtained including ES concentration of 0.01 mol/L, DT and BA concentrations of 0.1 mol/L, F/W of 0.25 and O/W of 1.0347, feeding in the middle stage at pH of 5.50. The ee(eq) of 0.98 and Y-eq of 0.99 can be achieved by symmetric separation with the number of stages of 32 under the optimal conditions. As S-ES is the desired product, 10 stages are enough to obtain 0.98 of ee(raffinate) for S-ES by asymmetric separation under the conditions containing ES concentration of 0.01 mol/L, DT and BA concentrations of 0.1 mol/L, F/W of 0.25 and O/W of 1.64, feeding in the Stage 2 at pH of 5.50.