A controllable strategy to fabricate the polymeric nanowires with high throughput and low cost is developed by using the thermal nanoimprint lithography (NIL) technique and self-assembled anodic aluminum oxide (AAO) template. The length of polymeric nanowires can be controlled by adjusting the duration of thermal NIL. A fill mechanism of thermoplastic intermediate polymer stamp (IPS) polymer pressed into the AAO nanopores is closely studied. The as-prepared IPS polymeric nanowire-based Surface-Enhanced Raman Scattering (SERS)-active substrate exhibits a remarkable reproducibility. The effective adsorption of the R6G as probe molecule near to hotspots generated at 3D vertically aligned polymeric nanowire SERS active substrates shows extraordinary enhancement of Raman signal with an enhancement factor (EF) of 105–106. The present strategy is of great guiding significance to broaden the use of thermal NIL technique and AAO template for the fabrication of other nanomaterials, especially for the flexible and transparent polymer-based nanomaterials.