The increasing concentration of heavy metals in the water ecosystems has become a noteworthy concern. This has accounted for various diseases due to the bioaccumulation in the human body. We propose a biocompatible, non-toxic technique for the identification of heavy metal ions from the contaminated aquatic samples. A urease biosensor, having interpenetrating network of Katira-cl-Acrylic Acid (AAc)/Polyaniline (PANI) hydrogel composite deposited onto the indium tin oxide (ITO). Enhanced hydrogel conductivity due to the incorporation of PANI has been gained by the interfacial polymerization of katira gum and aniline monomer in the ratio of 2:1 using N,N’-MBA and APS as cross-linker and initiator respectively. Structural, morphological, and electrochemical characterization of PANI/ITO, Katira-cl-AAc/PANI/ITO and Urease/Katira-cl-AAc/PANI/ITO electrode conducted using SEM, FT-IR, TGA, UV spectrometry, and cyclic voltammetry. A comparative study of different concentrations of cadmium, cobalt and mercury, has been done. The inhibition rates were found to be in the order of Hg2+ > Cd2+ > Co2+. Enzyme inhibition observed in the concentration range from 1.8 to 5 mM, 0.1 mM to 2 mM and 0.1 mM to 5 mM respectively. Urease/Katira-cl-AAc/PANI/ITO, exhibits greater stability, enhanced sensitivity and wide-range of detection limit (200 to 270 ng L-1) with a shelf life.