Electrochemical biosensors were used to develop a numerical model of transient mass transfer coupled with enzymatic reactions in thin multilayer microstructures. A finite volume method integrates the partial derivative mass-balance equations making the model very versatile. With a multilayer physical description, general nonlinear enzymatic kinetics and efficient space discretization, the model could be applied to various devices under a broad range of physicochemical conditions. Theoretical results were validated according to experimental data obtained with three different biosensors involving homogeneous or heterogeneous enzymatic catalyses. Model predictions are further discussed for a bienzymatic immobilized enzyme system. The biosensor transient behavior depended strongly on the location of the enzymes in the device and on the mass-transport features. A transient maximum on the current-time curves was predicted accurately by the model. This could be used to improve the biosensor performance. The model can also be a useful framework for designing new electrochemical microreactors.