Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. We have used layer-by-layer self assembly to fabricate bioelectronic interfaces containing the polyelectrolytes polyethylenimine (PEI) and polyacrylic acid (PAA) functionalized with dehydrogenase enzymes and their cofactors. The interfaces are able to achieve mediated electron transfer between the enzyme and the underlying electrode. The effect of the number of layers of PEI, mannitol dehydrogenase (MtDH), PAA, and toluidine blue O (TBO) on the performance of the bioelectronic interface was studied using cyclic voltammetry, chronoamperometry, constant potential amperometry, and electrochemical impedance spectroscopy. The current generated by the interface increased initially with the number of layers, and then reached a limiting value. As the number of layers increased, the calculated surface coverage increases 1-fold with each additional layer, but the apparent electron transfer coefficient and turnover rate decreased. A mathematical model of the multilayered bioelectronic interface has been developed and used to interpret the experimental results.