Nanotubes of cytochrome C (cyto-c) with glutaraldehyde (GA) or PSS based on the layer-by-layer (LbL) assembly through covalent binding and electrostatic adsorption have been fabricated. The combination of the template method and the LbL method for fabrication of nanotubes exhibits low cost, simplicity, and versatility. The tubular morphology of the assembled glutaraldehyde and cytochrome C film was demonstrated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) measurements. The components of the tubes were determined by energy-dispersive X-ray spectra (EDAX). It is found that the assembled tubes keep the proteins' biochemical activity and electronic activity by cyclic voltammograms. The measurements of ultraviolet spectra and circular dichroism (CD) on the assembled nanotubes confirmed the cyto-c existence in the tubes.