Nanofluidics show great promise for the control of small volumes and single molecules, especially for biological and energy applications. To build up more and more complex nanofluidics systems, a versatile and reproducible fabrication technique with nanometer precision alignment is desirable. In this article, two e-beam lithography methods to fabricate nanofluidic channels based on hydrogen silsesquioxane, a high-resolution negative-tone inorganic resist, are presented. The robustness and versatility of the fabrication processes are demonstrated on silicon, glass, and flexible substrates. The high precision ability is illustrated with nanometric alignment of nanofluidic channels on gold nanoparticles and nanotransistor sensors, as well as for 3D nanofluidics prototyping. Furthermore, an unexpected extremely slow water evaporation rate (≈1 week for 300 μm long nanochannels) is noticed. This feature enables a simple and reliable manipulation of nanofluidic chips for various studies.