Poly(o-anisidine) (POA) films were synthesized on mild steel from an aqueous oxalic acid solution by electrochemical polymerization of o-anisidine using cyclic voltammetry. These films were characterized by cyclic voltammetry, UV–visible absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The enzyme glucose oxidase (GOx) was entrapped into the POA film by a physical adsorption method. The resulting POA–GOx films were characterized by UV–visible absorption spectroscopy, FTIR and SEM. The amperometric response of the POA–GOx films was measured as a function of glucose concentration in phosphate buffer solution (pH 7.3). The POA–GOx films exhibited a fast amperometric response (1–5 s) and a linear response in the range of 2–20 mM glucose. The maximum current density and Michaelis–Menten constant of POA/GOx films were found to be ∼406 µA cm−2 and 1.03 mM, respectively. The shelf stability, operational stability and thermal stability of these films were also investigated.