Advancements have been achieved mainly on fabricating SERS substrates with sharp size distribution 27, 28, 29, in order to provide consistent SERS enhancements. Thus, one major challenge in the field is to improve the reproducibility. Yet such random aggregates lead to unequal enhancement factors and fluctuating SERS signals 26. The salt-induced aggregation of silver nanoparticle (AgNP) colloids is the most accessible method to form SERS active gaps 23, 24, 25. The SERS activity arises from the induced strong electromagnetic field confined in the junctions between plasmonic nanostructures, known as “hotspots” 21, 22. Hence, there is an urgent need to customize the SERS approach to better characterize protein structures at low concentration for greater biological significance. Besides, practical difficulties emerge when using conventional nanoparticle-based SERS substrates generated from salt-induced random aggregations to probe proteins in dilute solutions, such as low efficiency, poor reproducibility, and potential structural perturbation during long incubation time in SERS measurements 19, 20. In particular, the SERS study on alpha-synuclein, an IDP closely related to Parkinson’s disease 15, 16, 17, is scarce 18. However, the transient nature and the low population of oligomers make it challenging to characterize their structural features, which are responsible for the cellular toxicity and the on-going amyloid aggregation 14. The dynamic conversion from monomers to oligomers is the key step in the early pathological development 13. IDPs lack stable secondary and tertiary structures as monomers in aqueous environments, but sometimes self-assemble into oligomers with various structures and further grow into amyloid fibrils 11, which are associated with the incurable neurodegenerative diseases 12. It is also a powerful tool to characterize the dynamic ensembles of variable conformations of intrinsically disordered proteins (IDPs) 7, 8, 9, 10. Inheriting these advantages, surface-enhanced Raman spectroscopy (SERS) boosts up the sensitivity to detect proteins at low concentrations, even at the single-molecule level, to mimic physiological conditions 4, 5, 6. Raman spectroscopy probes the endogenous vibrations of molecules upon irradiation to delineate their chemical structures and surrounding environments 1, feasible for the label-free characterization of biomolecules in aqueous environments 2, 3. Hence, this SERS platform holds the promise to resolve the structural details of dynamic, heterogeneous, and complex biological systems. Moreover, it directly identifies the structural features of the transient species of alpha-synuclein among its predominant monomers at physiological concentration of 1 μM by reducing the ensemble averaging. This dynamic SERS detection window is placed in a microfluidic flow chamber to detect the passing-by proteins, which precisely characterizes the structures of three globular proteins without perturbation to their native states. Here, we develop an in situ optical tweezers-coupled Raman spectroscopy to visualize and control the hotspot between two Ag nanoparticle-coated silica beads, generating tunable and reproducible SERS enhancements with single-molecule level sensitivity. However, it is challenging to identify protein structures at low concentrations, especially for the proteins existing in an equilibrium mixture of various conformations. Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful tool to detect biomolecules in aqueous environments.