We describe the factors affecting the electron transfer process between the different components of a self-assembled mixed monolayer. The system is comprised of mixed monolayers containing aminoalkanethiols (AMATs) and ferrocenylalkanethiols (FATs) of variable chain lengths. We study the effects of different ratio of the two mixed monolayer components on the permeability of the monolayer towards a Ru(NH3)6C13 redox probe. In order to study the electrical communication between the enzyme and the mediator molecules, the enzyme glucose oxidase (GOx) was attached to the AMAT sites to create a biosensor device. The relative efficiency of a biosensor of each chain-length combination of FAT and AMAT was examined. In light of this comparison, we consider the critical factors for efficient electron transfer between the ferrocene mediator and the GOx redox active site immobilized as part of the surface-confined system. We find that the biosensor response is greatest when the enzyme and the FATs are attached to the surface with different alkane chain lengths. We also find strong evidence for the existence of domains of FAT and AMAT in the mixed monolayer system.