Multilayers of hemoglobin were uniformly deposited on a pyrolytic graphite electrode surface through a layer-by-layer self-assembly approach. The well-known anionic polyelectrolyte, DNA, was used to attract cationic hemoglobin due to the strong electrostatic interaction between DNA and the protein. Interestingly, the electron transfer reactivity of hemoglobin was significantly improved in the film, which in turn facilitated the electrocatalytic reduction of nitric oxide.