The range of analytes for which biosensors can be developed can be increased substantially by coupling multiple enzyme activities into reaction pathways. The sensitivity of the biosensors can also be increased (or amplified) dramatically by incorporating a substrate-recycling scheme. For low concentrations of substrate, this paper presents a theoretical model for a bi-enzyme biosensor that achieves signal amplification via substrate recycling. The bi-enzyme electrode was fabricated by co-immobilizing two enzymes: tyrosinase and NEST (neuropathy target esterase domain). The model was validated by assembling the bi-enzyme interface on a rotating disk electrode and measuring the biosensor's response to phenyl valerate substrate under varying rotating speeds. The model can help quantify the influences of mass transport, partition coefficients, enzymes, and electron-transfer kinetics on the metrological characteristics of the bi-enzyme electrode. This information can help optimize the performance of biosensors that use substrate recycling. (C) 2010 Published by Elsevier B.V.