Fabrication of a more superior nitrate potentiometric biosensor than previously achieved with NaR and NADH has been accomplished by co-entrapment of redox mediators and NaR into polypyrrole (PPy) film during galvanostatic polymerization of pyrrole. The replacement of NADH with redox mediators such as thionin acetate (ThAc), safranin (Saf), and azure A (AzA) gave more sensitive potentiometric responses, better minimum detectable concentration, linear concentration range and response time for nitrate than possible with NADH. The co-entrapment of ThAc, Saf, AzA and methyl viologen (MV) with NaR into PPy films also improved the Nernstian behavior of the electrode process beyond the capability of the PPy-NaR-NADH biosensor. Substantial reduction in volume and quantity of cofactor/mediator and, hence cost, was achieved by the replacement of NADH with a redox mediator. Only 50 μM of AzA was required to form a PPy-NaR-AzA biosensor which gave the most sensitive potentiometric response for nitrate, achieving a minimum detectable concentration of 10 μM, a linear concentration range of 50-5000 μM and a response time of 2-4 s.