Detection of low-concentration and diluted target analytes within specific hotspot regions is essential for ultrasensitive surface-enhanced Raman spectroscopy (SERS) analysis. However, achieving high reproducibility and sensitivity for SERS substrates remains a significant challenge. In this study, we present the fabrication of Ag nanoparticles (AgNPs) assemblies on a cylindrical plasmonic copper (Cu) rod substrate, using a drop-casting method to construct colloidal-based droplet-SERS platforms. Systematic optimization of droplet volume, analyte-to-nanoparticle ratio, and reaction conditions revealed the ideal parameters for performance enhancement. The cylindrical substrate exhibits sufficient hydrophobicity, confining the analyte to the tip of the SERS surface and preventing aqueous sample spreading, a common limitation in conventional substrates. This design enabled the analysis of samples as small as 4 μL, without requiring a drying step. Additionally, aligning the cylindrical substrates into a 3D-printed holder facilitated the production of an array for high-throughput analysis of aqueous samples. This strategy demonstrated outstanding SERS performance, with a relative standard deviation of 8.6% and substrate reusability. Under optimized conditions, Rhodamine B, a carcinogenic dye, was detected at a low threshold of 10–9 M (1 nM) in aqueous and real-world samples, including cotton candy without spiking. This application highlights the potential of this method for food safety analysis by addressing critical concerns regarding toxic contaminants, particularly those that pose health risks to children, who are the primary consumers of these products.