The chiral vicinal C–O/C–N bifunctional groups generated from enzymatic hydrocyanation represents a useful methodology. However, construction of the pharmacophore of β₂-adrenoreceptor agonists with this method remains a great challenge because of complete racemization of the benzylic alcohol during deprotection of the acetal groups. In this study, structure-guided redesign of a hydroxynitrile lyase originating from Prunus communis (PcHNL5) enables a highly enantioselective hydrocyanation of rigid benzo-ketal aldehyde which was proved to be resistant against racemization during the deprotection step, with dramatically improved productivity (>95% conversion vs <1%). X-ray structure analysis and kinetic study revealed the side chain of L331 tunes the substrate adaptability of bulky and rigid benzo-ketal aldehyde, thereby facilitating the formation of a series of valuable unnatural cyanohydrins in high enantiopurities and good yields. Furthermore, the HNL variant L331A was successfully applied for a gram-scale chemo-enzymatic synthesis of (R)-salmeterol, a long-term β₂-adrenoreceptor agonist, in an optically pure form (>99% ee) with an overall yield of 54%, which is the highest value reported.