Direct electron transfer of immobilized copper, zinc-superoxide dismutase (SOD) onto electrodeposited nickeloxide (NiOx) nanoparticle modified glassy carbon (GC) electrode displays a well defined redox process with formal potential of _0.03 V in pH 7.4. Cyclic voltammetry was used for deposition of (NiOx) nanoparticles and immobilization of SOD onto GC electrode. The surface coverage (G) and heterogeneous electron transfer rate constant (ks) of immobilized SOD are 1.75_10_11 molcm_2 and 7.5_0.5 s_1, respectively. The biosensor shows a fast amperometric response (3 s) toward superoxide at a wide concentration range from 10 mM to 0.25 mM with sensitivity of 13.40 nAmM_1cm_2 and 12.40 nAmM_1 cm_2, detection limit of 2.66 and 3.1 mM based on anodically and cathodically detection. This biosensor exhibits excellent stability, reproducibility and long life time.