A comparison of the analytical performances of several enzyme biosensor designs, based on the use of different tailored gold nanoparticle-modified electrode surfaces, is discussed. Glucose oxidase (GOx) and the redox mediator tetrathiafulvalene were coimmobilized in all cases by crosslinking with glutaraldehyde. The biosensor designs tested were based on the use of (i) colloidal gold (Au_coll) bound on cysteamine (Cyst) monolayers self-assembled on a gold disk electrode (AuE) and (ii) glassy carbon electrodes (GCEs) modified with electrodeposited gold nanoparticles (nAu). The results obtained with these designs were compared with those provided by a GOx/Cyst–AuE and a GOx/MPA–AuE. In the second case (ii), configurations based on direct immobilization of GOx on nAu (GOx/nAu–GCE) or on Cyst or MPA self-assembled monolayers (SAMs) previously bound on gold nanoparticles (GOx/Cyst–nAu–GCE or GOx/MPA–nAu–GCE, respectively) were compared. The analytical characteristics of glucose calibration plots and the kinetic parameters of the enzyme reaction were compared for all of the biosensors tested. The GOx/Au_coll–Cyst–AuE design showed a sensitivity for glucose determination higher than that achieved with GOx/Cyst–AuE and GOx/Au_coll–Cyst/Cyst–AuE and similar to that achieved with GOx/MPA–AuE. Moreover, the useful lifetime of one single GOx/Au_coll–Cyst–AuE was 28 days, remarkably longer than that of the other GOx biosensor designs.