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The droplet-based biphasic reaction is an efficient strategy for the fabrication of surface-bound nanostructures. Here, we developed a process of fabricating ordered micro-ring arrays of silver (Ag) nanostructures from surface nanodroplet reaction on a micro-patterned hydrophobic substrate for reproducible detection by surface-enhanced Raman spectroscopy (SERS). Our process consisted of the generation of surface nanodroplet arrays, followed by a biphasic chemical reaction between droplets and the continuous flow of silver nitrate (AgNO3) precursor solution. The parameters in the formation and reaction of the droplet array were well controlled to maintain the uniformity of Ag nanostructures throughout the substrate. By scaling up the process parameters and the size of the microchamber, we were able to produce a SERS substrate with a surface area of > 60 cm^2 in a single run. Such a large area could be sufficient for analyzing more than a thousand samples. We demonstrated the repeatability of SERS measurements using Ag nanostructures by analyzing three environmental (rhodamine 6G, chlorpyrifos, triclosan), a biological (indoxyl sulfate), and a psychoactive drug (tetrahydrocannabinol) compounds. 2D mapping of SERS intensities was also performed for both small and large-scale substrates by collecting data from more than 100 locations on the substrate. Our work demonstrated droplet-based biphasic reaction as a simple approach for the fabrication of SERS substrate with a large area. The technique may help to eliminate the requirement for sophisticated equipment for the fabrication of SERS active substrate.