We report a nanoimprint lithography method combined with photolithography on a bi-layer polymer setup to define nano-worms laterally in nano-gratings and produce uniform worm-shaped polymeric nanoparticles in aqueous solution by dissolving water soluble sacrificial layer poly(vinyl alcohol) (PVA). Process control of the thin residue layer, SU-8 curing using broadband UV source, and warm development to remove any residue are necessary to ensure success of this technique. The use of water soluble PVA as a releasing layer and elimination of an invasive plasma etching for the releasing process makes this protocol highly compatible with biomaterials. Direct release and suspension of fluorescent worm-shaped nanoparticles (length to width ratio up to 75) in aqueous solution were demonstrated. Compared to the worm-shaped nanoparticles made by self-assembly, these lithographically defined nano-worms have much better controllability and uniformity on the shape, size, and aspect ratio. The availability of these precisely defined non-spherical particles would be important to develop a comprehensive understanding of the shape effects of nanoparticles on their efficacy in nanomedicine applications.