The co-entrapment of Aspergillus niger-nitrate reductase (NaR) and azure A (AzA), as a redox mediator has been investigated for fabrication of a polypyrrole (PPy)-based electron mediated amperometric nitrate biosensor. Optimum formation of PPy-NaR-AzA film was achieved in a monomer solution which contained 0.3M Py, 0.1M KCl, 1U/mL NaR, 50μM AzA with an applied current density of 0.5mA/cm2 for 240s. The optimum applied potential of -0.25V for amperometric detection of nitrate with PPy-NaR-AzA biosensor was 50, 100 and 150mV more positive than those achieved with the use of safranin O (Saf), methyl viologen (MV) and bromophenol blue (BrB) or red (BrR), respectively, as mediators. Similar optimum applied of -0.25V was achieved with thionin acetate (ThAc) which was in the same phenothiazine group of mediators as AzA. The electron mediated amperometric detection of nitrate with the PPy-NaR-AzA biosensor also gave the best performance with a sensitivity of 40.8nA μM-1 cm-2, detection limit of 0.5μM, a response time of 18-20s and a linear concentration range of 20-500μM.