The proposed work aims to develop a sensitive surface-enhanced Raman spectroscopy (SERS) nano-biosensor. The inverted nano pyramid array on silicon substrate fabricated using electron beam lithography (EBL) was utilised as a master template and the mold was later replicated via nanoimprinting process to prepare gold-coated polymer nanopyramid three-dimensional (3D) SERS substrate. The fast and versatile replication process using nanoimprinting lithography (NIL) can produce polymer nanopyramids in a low-cost and reproducible fashion. Also, the proposed fabrication protocol can be easily upscale for large scale fabrication. The intense electric field confinement at nanotips and four edges of gold-coated polymer nanopyramid enhanced the Raman signal of probe molecules, i.e., Rhodamine 6G with a limit of detection down to 3.277 × 10⁻⁹ M was achieved. This work also underlines the efficiency of gold-coated polymer nanopyramid arrays in the spectral detection of hemoglobin proteins at low concentrations. The Raman signal enhancement mechanism was further studied through the electromagnetic simulation using COMSOL Multiphysics. In addition, bending test experiments were performed to understand the effect of flexibility on SERS signal response. The fabricated gold-coated polymer nanopyramids arrays could pave the way for the development of low-cost SERS platforms for the detection of hazardous biological and chemical compounds at ultra-low concentrations in practical applications.