A novel amperometric nitrite sensor was developed based on the immobilization of hemoglobin/colloidal gold nanoparticles on a glassy carbon electrode by a titania sol-gel film. The sensor shows a pair of well-defined and nearly reversible cyclic voltammogram peaks for Hb Fe(III)/Fe(II) with a formal potential (E°′) of −0.370 V, and the peak-to-peak separation at 100 mV s−1 was 66 mV vs. Ag/AgCl (3.0 M KCl) in a pH 6.9 phosphate buffer solution. The formal potential of the Hb Fe(III)/Fe(II) couple shifted linearly with pH with a slope of −50.0 mV/pH, indicating that electron transfer accompanies single-proton transportation. The sensor exhibited an excellent electrocatalytic response to the reduction of nitrite. The reduction overpotential was 0.45 V below that obtained at a colloidal gold nanoparticles/TiO2 sol-gel film-modified GCE. The linear range for nitrite determination for the sensor was 4.0×10−6 to 3.5×10−4 M, with a detection limit of 1.2×10−6 M. The stability, repeatability and selectivity of the sensor were also evaluated.