A layer-by-layer deposition strategy for preparing protein nanotubes within the pores of a nanopore alumina template membrane is described. This method entails alternately exposing the template membrane to a solution of the desired protein and then to a solution of glutaraldehyde, which acts as cross-linking agent to hold the protein layers together. The number of layers of protein that make up the nanotube walls can be controlled at will by varying the number of alternate protein/glutaraldehyde cycles. After the desired number of layers have been deposited on the pore walls, the alumina template can be dissolved to liberate the protein nanotubes. We show here that glucose oxidase nanotubes prepared in this way catalyze glucose oxidation and that hemoglobin nanotubes retain their heme electroactivity. Furthermore, for the glucose oxidase nanotubes, the enzymatic activity increases with the nanotube wall thickness.