Galvanostatic immobilization of nitrate reductase (NaR) with and without β-NADH, a biologically active form of nicotinic acid employed as a co-factor, into conducting polypyrrole (PPy) films has been successfully used to fabricate a novel potentiometric nitrate biosensor. The co-incorporation of NADH and NaR into PPy films resulted in substantial enhancement of the nitrate response compared to when present in solution. The formation of optimum PPy-NaR-NADH film for nitrate detection was accomplished with an applied current density of 0.5 mA cm-2, polymerization time of 4 min, 0.1 M pyrrole, 1 U/mL NaR, 0.3 M KCl and 0.3 mM NADH. Optimum potentiometric response for nitrate was obtained in 0.1 M phosphate buffer (pH 7.30). A minimum detectable concentration of 15 μM and a linear concentration range of 100-5000 μM were achieved with the nitrate biosensor, with a relative standard deviation of 1.9% (n = 6).