2-Arylpropionic acid derivatives, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of 2-arylacrylic acid derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction of 2-arylacrylic acid derivatives by either ERED wild-types or variants resulted solely in nonpharmacological (R)-enantiomers as the products. Here, we present the engineering of Saccharomyces pastorianus old yellow enzyme 1 (OYE1) into (S)-stereoselective enzymes, which afford pharmacologically active (S)-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro-S model of OYE1, the key residues of OYE1 that switch its stereoselectivity to an (S)-stereopreference were identified. Systematic site-directed mutagenesis screening at these positions successfully provided the (S)-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active (S)-derivatives including (S)-ibuprofen and (S)-naproxen methyl esters with up to >99% ee values. Moreover, the key residues and mutation strategy obtained from OYE1 could be further transferred to OYE 2.6 (from Pichia stipitis) and KnOYE1 (from Kazachstania naganishii) to create the (S)-stereoselective EREDs. Our results may provide a generalizable strategy for stereocontrol of OYEs and set the basis for biocatalytic production of (S)-profens.