A fast, economic, reproducible, accurate, effective, rugged and selective chiral-HPLC method was developed and validated for the enantiomeric resolution of nebivolol enantiomers [(+)-RRRS and (−)-SSSR)] in dosage formulation. The method was rapid as chiral separation occurred within only 12 min. The mobile phase used was n-heptane–ethanol–DEA (85:15:0.1, v/v) at 3.0 mL/min flow-rate with 225 nm detection. The column used was an amylase-based 3-AmyCoat (150 × 46 mm) [tris-(3,5-dimethylphenyl carbamate)]. The capacity factors of (+)-RRRS and (−)-SSSR enantiomers were 7.85 and 10.90 while the separation and resolution factors were 1.39 and 1.83, respectively. The limits of detection and quantitation for (+)-RRRS enantiomer were 4.5 and 10.00 µg/mL, while these values for (−)-SSSR enantiomer were 4.1 and 8.2 µg/mL, respectively. The linearity was observed in the concentrations range of 0.10–1.0 mg/mL for both enantiomers. The π–π interactions, hydrogen bonds, dipole–dipole interactions and steric effects control the chiral resolution of nebivolol enantiomers on the reported chiral column. The reported method can be used for the quality control of nebivolol in pharmaceutical preparations with good economy. In addition, this method can also be used for the analysis of (+)-RRRS and (−)-SSSR) enantiomers in biological and environmental samples. Copyright © 2011 John Wiley & Sons, Ltd.