Abstract A new conductometric biosensor based on interdigitated electrodes (IDEs) has been developed for the detection of enzymatic substrates using gold nanoparticles (GNPs), synthesized bellowing the citrate process, with an average diameter of 23 nm and functionalized with urease using layer‐by‐layer technique. A detection limit of 100 µM of urea is obtained when cross‐linked urease is directly immobilized on top of the IDEs (interdigitated distance: 20 µm) whereas a detection limit of 2 µM is obtained when urease functionalized gold nanoparticles are deposited on the top of the IDEs. The use of gold nanoparticles allows the increase of the sensitivity of detection (from 10 µS/mM to 107 µS/mM) due to the decrease of the thickness of probed zone.