We studied the direct electron transfer (DET) between glucose and electrogenerated AuCl4- catalysed by glucose oxidase (GOx) and gold nanoparticles (AuNPs) by scanning electrochemical microscopy (SECM). Well-defined AuNPs were prepared and attached onto an insulating surface. Studying the current transients of a gold microelectrode held within a few microns from the surface revealed that the AuNPs interacted with the GOx and were crucial for the DET from the glucose. We investigated very carefully the effect of pH and the size of the AuNPs on electron transfer. AuNPs of 16, 40 and 80 nm diameter were applied. The kinetics of electron transfer was analysed by the Michael-Menten kinetic mechanism. Interestingly, we found that the fastest DET was exhibited by the 40 nm AuNPs at pH 3 and 5. At higher pH, electron transfer was better catalysed by the 80 nm AuNPs. This was rationalized by the effect of the pH on the enzymatic structure and the charge of the AuNPs.