We report electrochemical preparation and characterization of a new biosensor made of nanostructured titanium dioxide (nano-TiO2) particles and deoxyribonucleic acid (DNA). Thionin (TN) redox mediator was electrochemically deposited onto DNA/nano-TiO2 modified glassy carbon electrode (GCE). The X-ray diffraction analysis, atomic force microscope (AFM) and scanning electron microscope (SEM) were used for surface analysis of TN/DNA/nano-TiO2 film. In neutral buffer solution, TN/DNA/nano-TiO2/GCE biosensor exhibited excellent electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2) and oxygen (O2). The biosensor shows excellent analytical performance for amperometric determination of H2O2, at reduced overpotential (−0.2 V). The detection limit and liner calibration range were found to be 0.05 mM (S/N = 3) and 0.05–22.3 mM, respectively. In addition, determination of H2O2 in real samples was carried out using the new biosensor with satisfactory results. The TN/DNA/nano-TiO2/GCE showed stable and reproducible analytical performance towards the reduction of H2O2. This biosensor can be used as an amperometric biosensor for the determination of H2O2 in real samples.