The sensing element of biosensor is prepared by the immobilization of urease onto nano-structured polyaniline (PANi)-Nafion® (NSPN)/Au/Al2O3 synthesized by the chronopotentiometric technique. The sensitivity of lead ion at an amperometric urease based biosensor is obtained to be 743.5 µA ppm−1 for the linear dynamic range of 0.1–1.0 ppm Pb2+. The irreversible inhibiting kinetic models of the mercury and lead ions at the amperometric biosensors are proposed. The values of Kd (dissociation equilibrium constant of adsorbed type of enzyme and heavy metal ion) and kirr (irreversible inhibition reaction rate constant) for mercury and lead ions are obtained to be 0.45 ppm, 1.66 × 10−3 s−1, 8.67 ppm and 3.16 × 10−3 s−1, respectively. Compared with lead ion detection, the higher sensitivity to mercury ion of the biosensor is mainly caused by a higher inhibition efficiency (kirr/Kd).