We report an extremely sensitive and ultra-low-cost PVDF-based SERS substrate using an easy fabrication process that can be conducted in a laboratory and scaled up for industrial applications. Hydrophilic PVDF membranes were sequentially dipped in ethanol and aqueous sodium chloride solution, following which multi-shaped Ag nanoparticles were deposited on the surface of the membranes by suction filtration. The simple solvating pretreatment led to an approximately 188-fold enhancement in the SERS signal intensity of R6G. Using the hydrophilic PVDF-based SERS substrate, subnanomolar sensitivity for R6G was achieved using 3 μL of sample volume on a 3×3 mm 2 SERS substrate within 1 min detection time and without the need for concentration or drying processes. To demonstrate the feasibility of the developed SERS substrate as a sensor for bacterial spore detection, it was applied for the detection of the representative biomarker dipicolinic acid (DPA). The limits of detection of DPA and Bacillus spores were measured as 1 ppm and 5×10 3 spores/mL, respectively. The optimized configuration of low substrate area and wet micro-analyte volume makes the PVDF-based SERS substrate a simple and low-cost tool for the trace-level detection of small molecules in a rapid, sensitive, and high-throughput manner. • An extremely sensitive and ultra-low-cost PVDF-based SERS substrate is reported. • The solvating pretreatment of PVDF led to 188-fold enhancement in the SERS signal. • A SERS spectrum was achieved using 3 μL of wet sample on a 9 mm 2 SERS substrate. • LOD of B. subtilis spores was 5×10 3 spores/mL much lower than the infective dose. • An easy fabrication process can be done in a laboratory and scaled up for production.