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In this research, we developed electrochemical biosensor which was composed of hemoglobin (Hb)-DNA conjugate on nanoporous gold thin film (NPGF) for hydrogen peroxide (H2O2) detection. For the first time, Hb and DNA was conjugated as a sensing platform for uniform orientation of Hb on electrode. The newly developed Hb-DNA conjugate was designed to prevent Hb from aggregation on electrode. DNA hybridization of Hb-DNA conjugate and complementary DNA (cDNA) on NPGF electrode induced uniformly assembled biosensor. Furthermore, NPGF electrode fabrication method was introduced to the increment of the surface area. To confirm the conjugation of Hb-DNA conjugate, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ultraviolet-visible spectroscopy (UV-VIS) were used. Formation of the NPGF electrode was verified by scanning electron microscope (SEM). Atomic force microscopy (AFM) was operated for the confirmation of Hb-DNA immobilization on electrode. The electrochemical property of fabricated electrode was investigated by cyclic voltammetry (CV). Also, H2O2 sensing performance of fabricated electrode was investigated by amperometric i-t curve technique. This sensor showed a wide linear range from 0.00025 to 5.00 mM and a correlation coefficient of R2 = 0.9986. The detection limit was 250 nM. Proposed biosensor can be utilized as a sensing platform for development of biosensor.