Bionanocomposites of hydrogenase and viologen-functionalized carbon nanotubes (H2ase/V-MWNTs) were prepared and characterized by using infrared spectra and scanning electron microscope. Cyclic voltammograms revealed two couples of redox waves corresponded to the electron transfer processes of viologens and [4Fe-4S]2+/1+ clusters of hydrogenase. The current intensity was enhanced in the H2 atmosphere, which suggested that the present bio-nanocomposites could be used as heterogeneous bio-catalyst to catalyze reversible reaction between protons and H2.