A hydrogen peroxide (H2O2) biosensor based on DNA-hemoglobin (Hb) modified electrode is described in this paper. The sensor was designed by DNA and hemoglobin dropletting onto gold electrode surface layer by layer. The sensor based on the direct electron transfer of iron of hemoglobin showed a well electrocatalytic response to the reduction of the H2O2. This sensor offered an excellent electrochemical response for H2O2 concentration below micromole level with high sensitivity and selectivity and short response time. Experimental conditions influencing the biosensor performance such as, pH, potential were optimized and assessed. The levels of the RSD's (< 5%) for the entire analyses reflected the highly reproducible sensor performance. Using the optimized conditions the linearity for the detection of the H2O2 was observed from 10 to 120 mu M with the detection limit of 0.4 mu M (based on the S/N=3). (c) 2005 Elsevier B.V. All rights reserved.