We study synthesis of L-phenylserine catalyzed by the enzyme L-threonine aldolase in a slug-flow microfluidic system. Slug-flow arrangement allows for the continuous refilling of sparingly soluble substrate (benzaldehyde) into an aqueous reaction mixture. We identified suitable composition of an organic phase to provide stable slug-flow in a wide range of operational parameters. Solvent screening revealed that tert-butyl methyl ether (TBME) as the organic solvent provides the most friendly environment for L-phenylserine synthesis due to a low degree of enzyme deactivation and high benzaldehyde concentration in the reaction mixture. The effects of substrate concentrations, enzyme concentration, and dimethylsulfoxide (DMSO) concentration on the L-phenylserine concentration in the product stream were examined and proper reaction conditions were identified. Experimental results on the L-phenylserine diastereoselectivity demonstrate that the amount of syn-conformation of L-phenylserine increases with the reaction time. High syn- to anti- concentration ratio is achieved with 2-methyltetrahydrofuran (MeTHF) solvent in a system with long residence time and, finally, low syn- to anti concentration ratio is provided by toluene environment and short residence time. (C) 2016 Elsevier Ltd. All rights reserved.