Nanotubes made from a fibrillar protein, namely, collagen, were fabricated by a template-based method combined with layer-by-layer (LbL) deposition. The ability to incorporate collagen in LbL multilayered film was first demonstrated by in situ quartz crystal microbalance and ex situ ellipsometry on a flat model substrate, using poly(styrene sulfonate) (PSS) as polyanion. Collagen-based nanotubes were then fabricated by alternately immersing a polycarbonate membrane, used as template, in PSS and collagen aqueous solutions. Direct evidence for nanotube formation was obtained by dissolving the membrane and imaging the liberated (PSS/collagen) nanostructures by scanning electron microscopy and by transmission electron microscopy. The proposed strategy constitutes a practical alternative to electrospinning as it allows a very good control over the dimensions (outside and inside diameters and length) of the resulting nanotubes. Besides their fundamental interest, collagen-based nanotubes are useful nano-objects for the creation of new nanostructured biomaterials with numerous potential applications in the biomedical field.