Poly(thionine) (PTH) film was generated on the electrochemically activated glassy carbon electrode (GCE(ea)) by using the two‐step cyclic voltammetric scan. Scanning electron microscopy, infrared spectral analysis and electrochemical measurement were employed to characterize the modified electrode surface. Hydroxyl radicals, which were produced by the Fenton‐like reaction, could induce the effective oxidization of PTH under near‐neutral condition and cause the notable enhancement of the cathodic peak current (Ipc) during the potential cycling process. Due to the binding of copper ions with the ligands liberated from VB12 and the inferior catalytic ability of Co2+ for the generation of hydroxyl radicals, the addition of VB12 into the Cu2+−H2O2 system inhibited the oxidization of PTH and resulted in the decrease of the Ipc value. The cathodic peak current change was linear with the logarithm of the VB12 concentration in the range of 10 nmol L−1–100 μmol L−1 with a detection limit of 2 nmol L−1 under optimal conditions. The developed sensor displayed excellent analytical performance including high sensitivity, good selectivity, acceptable reproducibility and satisfactory stability. The VB12 content in the injection sample was measured and the recovery values were in the range of 92.0 %–102 %.