There is a wide range of applications for three-dimensional (3D) nanochannels in biomedical systems and fluidic control. In this article, a simple and versatile technique to create 3D nanochannels with width from 200 nm to 2 mu m is demonstrated using sequentially stacked reversal UV nanoimprint of SU-8. Its advantages include controllable channel profile, low pressure and temperature for imprints, and flexibility in designing 3D channels by stacking. In a typical reversal UV imprint, SU-8 is spin coated on a glass mold and then transferred onto Si substrates by an UV imprint process at low temperature of 55 degrees C, low pressure of 2 MPa, and UV exposure of 1-4 s. While reversal UV imprinting top SU-8 layer onto bottom SU-8 layer, the UV exposure and imprint sequence of the top SU-8 layer and its effect on channel profile control are investigated. It has been found that initially UV-cured top SU-8 layer is preferred for good channel profile control because UV-cured SU-8 layer is prevented from flowing down into bottom SU-8 layer. (c) 2006 American Vacuum Society.