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Abstract: Direct electrochemistry of hemoglobin (Hb) was observed at glassy carbon electrode (GCE) modified with undoped nanocrystalline diamond (UND) and Hb multilayer films via layer-by-layer assembly. UV–VIS absorbance spectroscopy, electrochemical impedance spectroscopy and cyclic voltammograms were employed to characterize the film. The results showed that the UND had the effect of enhancing the electron transfer between Hb and the electrode surface. Hb in the multilayer films maintained its bioactivity and structure. It also exhibited a good catalytic activity towards the reduction of H2O2. The reciprocal of catalytic current showed a linear dependence on the reciprocal of H2O2 concentration ranging from 0.5 μM to 0.25 mM with a detection limit of 0.4 μM. The apparent Michaelis–Menten constant was estimated to be 0.019 mM. [Copyright &y& Elsevier]