The report is on an electrochemical biosensor with remarkably improved sensitivity toward nitrite. In this strategy, positively charged gold nanoparticle (PCNA) is used in combination with multiwall carbon nanotubes (MWCNT) by electrostatic adsorption for fabricating PCNA/MWCNT films. Then hemoglobin (Hb) biocatalyst will easily be attached to the surface of the combination films aforementioned. After that, the Hb/PCNA films are immobilized onto the Hb/PCNA/MWCNT films through layer-by-layer assembly technique. The (Hb/PCNA)(2)/MWNT/GC electrode thus prepared exhibits enhanced electrocatalytic behavior to the reduction of nitrite at -0.10 V versus SCE in 0.05 M H(2)SO(4) solution. On condition of the low detecting potential and low pH, interference caused by direct electrochemical oxidation or oxidizable substances can be prevented. Therefore, the modified electrode shows fast response time, very high sensitivity, good selectivity and stability. The current response of the sensor increases linearly with nitrite concentration from a range of 3.6 x 10(-6) to 3.0 x 10(-3) M with a detection limit(S /N = 3) of 9.6 x 10(-7) M.