Enzyme‐Catalyzed Efficient Synthesis of Enantiomerically Pure ( <i>S</i> )‐ and ( <i>R</i> )‐n‐Butylphthalide

3-n-Butylphthalide (NBP) is an effective commercial drug for the treatment of acute ischemic stroke, with its S-enantiomer, (S)-NBP, demonstrating clinical superiority over (R)-NBP. However, the stereoselective synthesis of both enantiomers with high enantiomeric excess (ee) presents significant challenges. Herein, a novel enzymatic strategy for the efficient and highly stereoselective synthesis of (S)- and (R)-NBPs under mild reaction conditions is presented. Specifically, two carbonyl reductases, SmCR_K6 and SsCR_K1, are engineered to facilitate the asymmetric reduction of the prochiral aromatic ketone, 2-pentanoyl benzonitrile (1), followed by intramolecular cyclization to produce chiral NBPs. SmCR_K6 exhibits a catalytic activity of 0.46 Umg^- ¹ protein, which is 23-fold greater than that of its parent enzyme, SmCR_V4, and the ee value of (S)-NBP increases from 2% to 94% (S). The catalytic activity of SsCR_K1 (9.46 U mg^-1 protein) is fourfold higher than that of its parent, SsCR, and the ee of (R)-NBP reachs 99%. In the preparative synthesis, (S)-NBP and (R)-NBP are generated continuously in a 3D microfluidic reactor, achieving space-time yields that are 9-fold and 30-fold higher, respectively, than those obtained in batch reactions. This continuous-flow enzymatic process has the potential for future scale-up for the industrial production of these important chiral drugs and similar derivatives.