A nanocomposite-based sensor that responds selectively to mandelic acid (MA) enantiomers was constructed with Dexamethasone (DEX) as a chiral recognition element. The DEX-modified electrode was selective towards MA enantiomers compared with the glassy carbon electrode (GCE) modified by graphene (GR) nanosheets or bare GCE. The entrapment of DEX within the overoxidised polypyrrole (OPPy) film occurred on the surface of the GR-modified electrode using a two-step electrodeposition approach. The deposited film was characterized by scanning electron microscopy (SEM), differential pulse voltammetry (DPV), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The OPPy-DEX/GR/GCE sensor for sensing of MA enantiomers required similar to 0.3 V less overpotential than the GC electrode. Upon exposure to (S)- and (R)-MA, the sensor showed the current signals with different ratios, which were differentiated at the voltages of 1.38 and 1.43 V (vs. Ag/ AgCl), respectively. Under optimal conditions, the chiral sensor exhibited a good linear relationship with MA enantiomers concentrations ranging from 1 to 25 mM with a detection limit of 0.25 mM. Due to the highly overlapping signals, partial least squares (PLS) regression was applied to distinguish the MA enantiomers in synthetic mixtures. In addition, the genetic algorithm-based potential selection procedure was applied to optimize the number of PLS factors used for the building of the PLS calibration models, and also its predictive ability was studied.