This study described the bioelectrochemistry of hemoglobin (Hb) at multiwall carbon nanotubes (MWCNTs) noncovalently functionalized with biopolymers of sodium alginate (SA). The characteristics of Hb/SA–MWCNTs composite film were studied by using FT-IR spectroscopy, UV–vis spectroscopy and electrochemical methods. Hb immobilized on SA–MWCNTs composite film retained its native secondary structure, achieved direct electron transfer with the apparent heterogeneous electron transfer rate constant (ks) of (9.54±0.883)s−1 and showed excellent bioelectrocatalytic activity to the reduction of hydrogen peroxide. The amperometric response of the biosensor varied linearly with the H2O2 concentration ranging from 40 to 200μM, with a detection limit of 16.41×10−6M (S/N=3) and the good long-term stability. Finally, we applied this proposed method to investigate the concentration of hydrogen peroxide in real samples.