In conductometric enzymatic biosensors, enzymatic reaction is confined close to the interdigitated electrode surface, because enzyme is cross-linked in contact with this surface in the presence or absence of nanoparticles. The effect of the use of a new type of doubly-functionalized gold nanoparticles (PF-HEG-Au NPs) on the response of conductometric biosensor based on interdigitated electrodes (IDEs), for the detection of enzymatic substrates was studied. Gold nanoparticles (AuNPs) were first synthesized following the citrate process, with an average diameter of 14 nm. AuNPs were then functionalized with 11-mercaptoundecylhexaethyleneglycol (HEG) and then with 1H,1H,2H,2H-perfluorodecanethiol (PF). The doubly-functionalized AuNPs were characterized using TEM, UV-Vis spectrophotometry and FTIR spectroscopy. Urease, mixed with these doubly functionalized AuNPs, was then cross-linked with glutaraldhedyde vapor on the IDE surface. In the presence of urea, the conductometric response was measured in a differential mode. The best sensitivities for urea detection were obtained with PF-HEG-Au NPs (520 µS /mM and 0.5µM of detection limit), as compared to 284µS/mM and 2µM of detection limit with bare Au NPs, PF-AuNPs and HEG-AuNPs, and 1.07µS/mM and 100 µM of detection limit with urease directly crosslinked on IDEs.When stored in phosphate buffer (5 mM, pH 6.7) at 4 °C, the biosensor with PF-HEG-Au NPs showed good stability for more than 12 days.