Conducting polymer hydrogels (CPH) are a unique class of materials, which have both the advantageous features of hydrogels and organic conductors. Herein, we report a new urea biosensor in which the working electrode is modified by a CPH, specifically a composite made of conducting electro-polymerized aniline in the non-conducting hydrogel of polyacrylamide and polyvinyl alcohol for immobilization of the enzyme urease. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies depict the porous structure of the hydrogel, which favours the high density immobilization of enzymes. Fourier transform infrared spectroscopy (FTIR) studies confirmed the grafting of polyaniline within the matrix. Electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) and cyclic voltammetry (CV) predict high enzyme loading in the hydrogel. Bode plot analysis with the [FeCN₆]^3−/4− redox couple shows the partial capacitive behaviour of the bioelectrode in the frequency range of 10² to 10³ Hz. Factors, such as scan rate and pH of the electrolyte, were optimized to give the best results. DPV could detect 1.5–1000 μM urea with the lowest detection limit (LOD) of 60 nM and sensitivity of 878 μA mM⁻¹ cm⁻². The relatively low Michaelis–Menten constant (K_m) value of 113 μM indicates the high affinity of the enzyme for urea. EIS depicts an increase in charge transfer resistance (R_CT) with an increase in urea concentration (LOD = 14 μM). The proposed sensor exhibits a very good performance with respect to detection limit, sensitivity, K_m value, preparation method, cost, sample preparation, stability, selectivity and reproducibility. This sensor was successfully applied for the detection of urea in milk, puffed rice, soil and human blood. Statistical analysis of the results shows good agreement with commercial spectrophotometric analysis.