Miniaturized flow reactors with immobilized biocatalysts offer enormous potential for process intensification. They enable long-term use of biocatalysts, continuous operation that significantly outperforms batch processes, and efficient mass and heat transfer that results in highly controlled reaction conditions. Despite their increasing use in biocatalytic processes, optimization of reactor design and operating conditions based on mathematical description is very rare. This work aims to fill this gap by developing and validating a mathematical model for the continuous biotransformation process in a microreactor between two plates with immobilized whole cells in hydrogel layers on the bottom and top of the reactor. A biocatalytic production of l-malic acid by fumaric acid hydration using permeabilized Saccharomyces cerevisiae whole cells was used as a model reaction. The diffusivity of substrate and product in a liquid phase and in a copolymeric hydrogel layer and the reaction kinetic parameters considering the Michaelis-Menten kinetics of the reversible enzymatic reaction were estimated in initial batch experiments. The results obtained in a continuously operated microbioreactor with immobilized whole cells at different fumaric acid concentrations and flow rates were in excellent agreement with the predictions of the developed mathematical model comprising transport phenomena and reaction kinetics. Based on the validated model and using time-scale analysis with characteristic times, the optimal process and operating conditions for the developed microbioreactor system were determined. The model predicts an equilibrium conversion of fumaric acid at the highest inlet concentration tested when using a liquid height of 200 μm and a hydrogel thickness on both sides of the channel of 400 μm at a residence time of 30 min.