Poly(o-phenylenediamine) (PoPD) film has been electrochemically prepared on Pt electrode in an acetonitrile–water medium containing o-phenylenediamine (oPD) monomer and (±)-10-camphorsulfonic acid (HCSA) by using the cyclic voltammetry (CV). The PoPD film (PoPD–CSA) has been characterized by FTIR, CV, EIS, FESEM, and conductivity measurement. The glucose biosensor (Pt/PoPD–CSA/GOx) has been prepared from the PoPD coated electrode by immobilizing glucose oxidase (GOx) enzyme using glutaraldehyde. The biosensor shows a low detection limit and wide linear working range, a good reusability, long-term stability, and anti-interference ability. The Pt/PoPD–CSA/GOx has possesses higher sensitivity (2.05 μA/mmol L−1) and affinity to glucose due to the use of CSA ion as dopant. The linear concentration ranges of Pt/PoPD–CSA/GOx have been found to be 9.6 × 10−3 to 8.2 mmol L−1 from calibration curve and 4.6 × 10−2 to 100 mmol L−1 from the relationship between the (1/glucose concentration) and (1/current difference). © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 39864.