Candida antarctica lipase B (CALB) is a versatile and robust lipase with high activity and enantioselectivity in the resolution of alcohols and amines. Poor specific activity toward carboxylic esters that are branched or substituted in close proximity to the carboxyl group limits the application of CALB. In this study, a semi-rational engineering approach was successfully applied to tailor CALB for efficient synthesis of the moxifloxacin chiral intermediate through enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate. The combinatorial active-site saturation test was performed on CALB and two key residue sites, 189 and 190, were identified in the determination of hydrolytic activity. The specific activity and catalytic efficiency (k_cat/K_m) of the best variant mut-I189K were increased by 286- and 193-fold, respectively, without impacting its enantioselectivity. With 0.1 g L⁻¹ of purified mut-I189K loading, 1 M substrate was asymmetrically hydrolyzed with 49.9% conversion within 5 h, resulting in a space–time yield of 583.8 g L⁻¹ d⁻¹. These results demonstrated that mut-I189K is a competitive biocatalyst for large-scale preparation of the moxifloxacin chiral intermediate.