Biocompatible poly(L-lactide) (PLA) was synthesized by a lactide monomer polymerization, with the structure be confirmed by FT-IR spectrum. The direct electrochemistry and electrocatalytic properties of Hemoglobin (Hb) and PLA co-assembled film (Hb-PLA film) cast on glass carbon (GC) electrode were investigated. A pair of well-defined redox peaks of Hb with a formal potential (E-0) of about -0.38 V (vs. SCE) in a pH 7.0 phosphate buffer solution (PBS) were obtained at the Hb-PLA film modified GC electrode. The apparent heterogeneous electron transfer rate constant (k(s)) was evaluated to be 42.3 s(-1) by the Laviron's equation. The surface concentration (Gamma*) of the electroactive Hb in the PLA film was estimated to be 4.39 x 10(-11) Mol cm(-2). Moreover, the Hb entrapped in PLA film showed good bioelectrocatalytic activity for the electrochemical reduction of H2O2. The amperometric response varied linearly with the H2O2 concentration ranged from 10 mu M to 120 mu M, with a detection limit of 12.3 x 10(-6) M and a high stability of the immobilized Hb in PLA film. Finally, we applied this proposed method to investigate the concentration of H2O2 in real samples. (C) 2008 Elsevier B.V. All rights reserved.