This paper describes an efficient and versatile method for the fabrication of nanostructured substrates from a piece of tendon which comprises aligned collagen nanofibers. We used a microtome to generate the tendon slices (10-50 µm thick), which were used as a scaffold for guiding directional cell growth. Highly aligned and uniform monolayer cells sheets were obtained. The tendon slices were used as a master, and the nanostructures outlined by the bundles of collagen nanofibers were successfully transferred onto a polystyrene film using standard soft lithography. The cell growing on the nanostructured polystyrene substrate showed good adhesion and alignment. The technique developed here enables one to fabricate nanostructured substrates without using any traditional micro/nanofabrication tools. The nanostructured substrate, e.g. a slice of tendon, has excellent biocompatibility and relatively good mechanical stability, which makes this technique useful in constructing complicated 3D tissues.