A metal-directed assembling approach has been,developed to encapsulate hydrogenase (H(2)ase) within a layer-by-layer (LBL) multilayer of carbon nanotube polyelectrolyte (MWNT-PVPMe), which showed efficient biocatalytic oxidation of H(2)ase. The MWNT-PVPMe was prepared via a diazonium process and addition reactions with poly(4-vinylpyridine) (PVP) and methyl iodide (MeI). The covalently attached polymers and organic substituents in the polyelectrolyte comprised 60-70% of the total weight. The polyelectrolyte was then used as a substrate for H(2)ase binding to produce MWNT-PVPMe@H(2)ase bionanocomposites. Xray photoelectron spectra revealed that the bionanocomposites included the elements of Br, S, C, N, O, I, Fe, and Ni, which confirmed that they were composed of MWNT-PVPMe and H(2)ase. Field emission transmission electron microscope images revealed that the H(2)ase was adsorbed on the surface of MWNT-PVPMe with the domains ranging from 20 to 40 nm. Further, with the use of the bionanocomposites as nanolinkers and Na2PdCl4 as connectors, the (Pd/MWNT-PVPMe@H(2)ase)(n) multilayers were constructed on the quartz and gold substrate surfaces by the Pd(II)-directed LBL assembling technique. Finally, the as-prepared LBL multilayers were used as heterogeneous catalysts for hydrogen oxidation with methyl viologen (MV2+) as an electron carrier. The dynamic processes for the reversible color change between blue-colored MV+ and colorless MV2+ (catalyzed by the LBL multilayers) were video recorded, which confirmed that the H(2)ase encapsulated within the present-LBL multilayers was of much stronger stability and higher biocatalytic activity of H-2 oxidation resulting in potential applications for the development of H-2 biosensors and fuel cells.