Single molecule detection using nanopores has proven to be a powerful way of identifying biomolecules. There-fore, a variety of materials and methods have been developed by the nanopore community to generate credible outcomes. Here we report as the first time a novel polyimide nanopore platform for single DNA sensing. Novelty of the platform arises both from the ultra-thin structure of the polyimide membrane and also the technique used in the nanopore fabrication on a polymer membrane. Furthermore, the ultra-thin nature of the membrane eliminates the involvement of highly demanding fabrication steps often employed in the solid-state nanopore membrane production. Herein we present the fabrication of nanopores in such an ultrathin polymer membrane and its successful application in sensing DNA molecules at single molecule level, holding a promise as a potential single-molecule analytical device unlike its common use in research.