One-dimensional protein nanostructures offer many advantages for biomedical applications. Rather than fabricate primary nanostructures with inorganic materials and then functionalize with proteins, it is desirable to develop a fabrication method to make nanostructures that are entirely protein. Fabrication of protein and polymer nanostructures is possible by layer-by-layer assembly within nanoporous templates. Typically these structures are composites of two or more materials. Few studies have demonstrated the fabrication of single component protein nanostructures using this method. In this paper, we report our effort toward the fabrication of single-component avidin nanotubes using a layer-by-layer electrostatic assembly method adapted from the literature. We investigated the use of two different template pretreatment methods to strengthen the attraction between the initial protein layer and our template. During our investigation, we revealed a significant flaw with the published works upon which our fabrication method was based which seriously compromised the legitimacy of the approach. As a result, we modified our initial method, and we are able to demonstrate the fabrication of glucose oxidase/avidin nanostructures using an electrostatic layer-by-layer assembly in conjunction with one of the template pretreatment methods we investigated.