Experimental and model‐based studies of acrylonitrile biohydration catalyzed by free cells were conducted in this work. The high ratios of the two phases of the reaction system and the effects of inhibition and inactivation on the enzyme make the process complicated; thus, its exact kinetics have never been reported. To fully understand and model the reaction, a microstructured chemical system was employed to investigate droplet dispersion, the mass transfer process, the intrinsic kinetics with inhibition and inactivation, and the preparation process of acrylamide. By adjusting the structure size and flow conditions of the microreactor, acrylonitrile droplets of 40 μm were obtained and enhanced mass transfer was achieved. Compared to previous studies, the microchemical system in our work provides more accurate kinetic parameters, with a much higher maximum reaction rate and much smaller Michaelis constant. The numerical predictions of the process model show good agreement with experimental results.