Anionic redox mediator-incorporated polymer scaffold on glassy carbon (GC) electrode has been used for the electrocatalytic determination of nitrite. The polymer based on branched polyethylenimine (BPEI) crosslinked with ethylene glycol diglycidyl ether (EGDE) (BPEI-EGDE) has been employed as the potent scaffold. BPEI-EGDE film on GC electrode has been used to immobilize anionic redox mediator [Fe(CN)(6)](3-) by ion-exchange and utilized as the scaffold for the electrochemical determination of nitrite. The voltammetric analysis confirms the confinement of the redox mediator inside the polymer film. This polymeric scaffold, GC/BPEI-EGDE/[Fe(CN)(6)](3-) exhibits a reversible redox response for [Fe(CN)(6)](3-/4-) and retains its redox activity and stability even in strong acidic conditions. The redox mediator confined into the polymer scaffold catalyzes the reduction of nitrite. Significant enhancement in the cathodic current associated with the concomitant decrease in the anodic current was observed in the presence of nitrite. The electrocatalytic response of the scaffold was exploited for the amperometric sensing of nitrite at the potential of 450 mV. The polymer scaffold is highly sensitive (28.5 A mu A mM(-1) cm(-2)) and it could detect nitrite at micromolar levels (detection limit is 4.8 A mu M). The scaffold has been successfully used for the real sample analysis and high selectivity, stability, and reproducibility have been achieved.