Based on immobilizing manganese oxide nanospheres (MnOxNsph)/deoxyribonucleic (DNA) on glassy carbon electrode (MnOxNsph/DNA/GCE), a new electrochemical biosensor for the detection of thiourea (TU) was fabricated. In order to prepare DNA template, cyclic voltammetry (CV) method was used. Scanning electron microscopy (SEM) was used for characterization of MnOx/DNA. Electrochemical impedance spectroscopy was utilized to confirm the successful stepwise assembly procedure of the biosensor. The electrocatalytical behaviors of the biosensor were also investigated by cyclic voltammetry and differential pulse voltammetry (DPV). The results showed that MnOxNsph/DNA exhibited a remarkable electrocatalytic activity for the oxidation of TU under optimal conditions. The linear range, detection of limit and sensitivity were calculated for oxidation peaks (OX1 and OX2). This electrode demonstrated many advantages such as high sensitivity, low detection of limit, excellent catalytic activity at natural pH values, remarkable antifouling property toward TU and its oxidation product for the two oxidation peaks. (C) 2017 Elsevier Inc. All rights reserved.