A chiral biosensing platform was developed using betamethasone (BMZ) as chiral recognition element through multilayered electrochemical deposition of BMZ, overoxidized polypyrrole, and nanosheets of graphene (OPPy-BMZ/GR), for enantio-recognition of mandelic acid (MA) enantiomers. The deposited film was characterized by scanning electron microscopy, differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy. It was shown that the chiral sensing platform can discriminate R- and S-MA differential pulse voltammetry signals, at the voltages of 1.35 and 1.33 V (vs Ag/AgCl), respectively. To tackle the problem of highly overlapping peaks of these enantiomers, the partial least squares (PLS) regression and genetic algorithm-PLS (GA-PLS) were used for simultaneous quantification of MA enantiomers. Generally, variable selection by genetic algorithm provided an improvement in prediction results when compared to full-voltammogram PLS. Good analytical performances were obtained despite the inherent complexity of the simultaneous determination.