Objective To enzymatically synthesize enantiomerically pure beta-amino acids from beta-keto nitriles using nitrilase and omega-transaminase. Results An enzyme cascade system was designed where in beta-keto nitriles are initially hydrolyzed to beta-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently beta-keto acids were converted to beta-amino acids using omega-transaminases. Five different omega-transaminases were tested for this cascade reaction, To enhance the yields of beta-amino acids, the concentrations of nitrilase and amino donor were optimized. Using this enzymatic reaction, enantiomerically pure (S)-beta-amino acids (ee > 99%) were generated. As nitrilase is the bottleneck in this reaction, molecular docking analysis was carried out to depict the poor affinity of nitrilase towards beta-keto acids. Conclusions A novel enzymatic route to generate enantiomerically pure aromatic (S)-beta-amino acids from beta-keto nitriles is demonstrated for the first time.