Continuous monitoring of the dynamic concentration of biomolecular compounds holds immense promise across multiple sectors, including healthcare, environmental management, food production, and drug development. Nevertheless, significant challenges persist in the advancement of biosensor technology, particularly regarding the continuous monitoring of ultralow concentrations directly in biological fluids. Here we detail the development of a novel biosensing platform based on a nanoparticle-on-film (NPoF) construct with single-molecule resolution. Interactions between single gold particles and a gold film are monitored with high signal-to-noise ratio owing to the resonant coupling between propagating and localized plasmons. The reversibility of the interactions provides for internal resetting, allowing for continuous biomolecular monitoring of ssDNA, micro RNA, and proteins. The combination of tailored surface chemistry and very small probe particles provides efficient mass transport and enables continuous monitoring at unprecedented sub-picomolar concentrations. We further introduce multicomponent kinetic fingerprinting that efficiently suppresses non-specific interactions and enables sub-picomolar continuous monitoring directly in full serum. In the future, the platform will enable the monitoring of species that are currently inaccessible due to their low concentrations and may find application in healthcare, environment and industrial settings.