A reagentless electrochemical chiral sensor for discriminating mandelic acid (MA) enantiomers was achieved by adsorbing hemoglobin (Hb) on the multiwalled carbon nanotubes (MWNTs) modified glassy carbon electrodes (Hb/MWNTs/GCE). The interactions between Hb and Fe(III) complexes with MA enantiomers were measured by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and ultraviolet-visible spectroscopy (UV-Vis). The results showed that Hb could recognize MA enantiomers in the presence of Fe(III), and larger response signals were obtained from S-MA. The influence factors including pH, response time, metal-ion species and the concentrations of MA enantiomers were studied on the effect of chiral sensing, and the possible mechanism for stereospecific interaction was explored. In addition, the sensor was employed in determining the enantiomeric ratio of MA enantiomer mixtures, in which the excellent performance with relative error less than 2.6% highlighted the acceptable accuracy. (C) 2013 The Electrochemical Society. All rights reserved.