In this paper, we demonstrate a novel method for high throughput patterning of bioprobes with nanoscale features on biocompatible polymer substrate. Our technique, based on nanostencil lithography, employs high resolution and robust masks integrated with array of reservoirs. We show that the smallest pattern size can reach down to 100 nm. We also show that different types of biomolecules can be patterned on the same substrate simultaneously. Furthermore, the stencil can be reused multiple times to generate a series of identical patterns at low cost. Finally, we demonstrate that biomolecules can be covalently patterned on the surface while retaining their biofunctionalities. By offering the flexibility on the nanopattern design and enabling the reusability of the stencil, our approach significantly simplifies the bionanopatterning process and therefore could have profound implications in diverse biological and medical applications. © 2012 American Chemical Society.