We developed soft lithography-based nano-well surface-enhancement Raman scattering (SERS) substrates with sub-1 pM detection sensitivity, which can be useful for label-free genomics, proteomics, or environmental monitoring biosensors. We batch-fabricated pillarlike nanostructures on a silicon wafer (i.e. master)  to replicate the complementary nanowell structures using polydimethylsiloxane (PDMS) by soft lithography, and deposited Ag thin film on the nanostructured PDMS. A glass substrate is used as an optical window and to form microfluidic channels integrated with the Ag/PDMS nano-well substrate. SERS spectra of the Rhodamine 6G (R6G) and adenosine molecules in microfluidic channels are detected on the nano-well SERS substrates with only 300 μW excitation power. The Raman scattering signal enhancement on the Ag/PDMS nano-well SERS substrate is more than 107 times higher than that on the smooth silver layer on PDMS. The present successful label-free detection of sub-picomolar level adenosine molecules is important to future studies of low concentration nucleic acid reactions such as hybridization, denaturization, nuclease, and transcription in microfluidics. The monolithic integration of the Ag/PDMS nano-well in microfluidics promises the label-free detection of singlemolecular level biochemical reactions in multiplexed aqueous environments.