Supramolecular hydrogels of conducting polymers are successfully used in bioelectrochemistry because of their mechanical and swelling properties of gels added to the specific electron transport properties of conducting polymers. We have studied polyaniline-poly(styrene sulfonate) (PANI-PSS) hydrogel as a substrate for the urease. The hydrogels were synthesized at pH = 0 and pH = 5. PANI-PSS hydrogel is a supramolecular self-assembly material consisting of positively-charged PANI chains and negatively-charged PSS chains. The hydrogel was studied by cyclic voltammetry, infrared and Raman spectroscopy and Scanning Electron Microscopy (SEM). Raman spectra revealed presence of phenazine rings in the hydrogel structure. Phenazine rings form covalent cross-linkers contributing to the hydrogel mechanical stability. The covalent cross-linkers influence the cyclic voltammetry responses of the hydrogel in acidic media. We tested the activity of urease immobilized in the PANI-PSS hydrogel by the physical adsorption or by the covalent bonding with the carbodiimide reaction. The enzyme immobilized in hydrogels prepared at higher value reveals significantly higher sensitivity. The method of the enzyme immobilization has smaller impact on the sensitivity. All hydrogel sensors reveal largely higher sensitivity to urea comparing to urease immobilized in the typical electrochemically deposited PANI films. The sensitivity of urease covalently bond to the hydrogel obtained at pH = 5 was as high as 1693 mu A/(mol dm(3)). The sensor response was linear in the urea concentration range from 10(-4) to 7 X 10(-2) mol/dm(3). (C) 2013 Elsevier Ltd. All rights reserved.