Characterizing a monolayer of biological molecules has been a major challenge. We demonstrate nanophotonic waveguide enhanced Raman spectroscopy (NWERS) of monolayers in the near-infrared region, enabling real-time measurements of the hybridization of DNA strands and the density of submonolayers of biotin–streptavidin complex immobilized on top of a photonics chip. NWERS is based on enhanced evanescent excitation and collection of spontaneous Raman scattering near nanophotonic waveguides, which for a 1 cm silicon nitride waveguide delivers a signal that is more than 4 orders of magnitude higher in comparison to a confocal Raman microscope. The reduced acquisition time and specificity of the signal allows for a quantitative and real-time characterization of surface species, hitherto not possible using Raman spectroscopy. NWERS provides a direct analytic tool for monolayer research and also opens a route to compact microscopeless lab-on-a-chip devices with integrated sources, spectrometers, and detectors fabricated using a mass-producible complementary metal oxide semiconductor technology platform.