Surface-enhanced Raman scattering (SERS) has recently been considered as one of the most promising tools to directly analyze small molecules without labels, owing to advantages in sensitivity, specificity, and speed. However, collecting reproducible SERS signals from small molecules on substrates or in solutions is challenging because of random molecular adsorption on surfaces and laser-induced molecular convection in solutions. Herein, we report a novel and efficient way to collect SERS signals from solution samples using three-dimensional nanoplasmonic wells spontaneously formed by interfacial reactions between liquid polydimethylsiloxane (PDMS) and small droplets of metal ion solutions (e.g., HAuCl4 and AgNO3). A SERS signal is easily maximized at the center near the bottom of the well due to spherical feature of the fabricated wells and electromagnetic field enhancement by the metallic nanoparticles (e.g., Au and Ag) integrated on their surfaces. Through the systematic control over the volume, concentration, and composition of the metal ion solution, optical functions of the nanoplasmonic wells were optimized for SERS, which was further amplified by exploiting the plasmonic couplings with colloidal nanoparticles. By using the optimized nanoplasmonic wells and the detection protocol, we successfully obtained intrinsic spectra of biomolecules (e.g., adenine, glucose, amyloid β) and toxic environmental molecules (e.g., 1,1'-diethyl-2,2'-cyanine iodide and chloromethyliothiazolinone/methylisothiazolinone) as well as Raman active molecules, such as rhodamine 6G and 1,2-bis(4-pyridyl)ethylene at a low concentrations down to the picomolar level. Our detection platform provides a powerful way to develop highly sensitive sensors and high-throughput analyzing protocols for fieldwork applications as well as diagnosing diseases.