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Single-molecule detection (SMD), which represents the detection limit in molecular spectroscopy, has opened a new research realm in the fields of catalysis, DNA sequencing and protein analysis. Meanwhile, it provides  new insights into the understanding of the molecule behaviors in a complex system. Specifically, SMD enables the quantitatively identifying of molecules accurate to single digit, provides the molecular distribution state under specific environments, and permits the in-situ observation of signal fluctuations of a single-molecule under chemical stimulus. Single-molecule surface-enhanced Raman spectroscopy (SM-SERS) is a new subject in SMD which features specific recognition of molecules by identifying the molecular chemical bonds. It is a non-destructive technology which reflects the vibration energy and rotational energy information of molecules. This technique employs metallic nanostructures to form surface plasmon resonances (SRP) under external excitation. The SPRs generate strong local electromagnetic fields ("hot spots") around metal surface to amplify the Raman signal of probe molecules in the vicinity of plasmonic materials. The giant field enhancement endows SERS superior sensitivity in trace molecule detection down to a single-molecule level. The SM-SERS offers a facile method to track the evolution of a single molecule, revealing the reaction pathways, adsorption state and distributions, and charge exchanges between the molecule and surrounding environment. Though SM-SERS has been proposed more than 20 years ago, the acquisition of SM-SERS spectra remains a bottleneck in this field due to the disability in judging the origins of these spectra. On the other hand, the lack of knowledge in analyzing SM-SERS spectra also limits the development of SM-SERS as the origins of molecule behavior at a micro level is basically unknown to the public. This review paper covers the development of SM-SERS, the past and current methods of verifying SM-SERS including the non-statistical and the bi-analyte statistical methods, the investigation into the understanding of the fluctuation characteristics of SM-SERS, as well as the related mechanisms with regard to the unique phenomena in SM-SERS such as molecule diffusion, spectral blinking and broadening. We hope this review can help the readers to relate the characteristics in SM-SERS with the origins of molecular variations during the detection, in this way to get a clear and in-depth understanding of the roadmap for SM-SERS.

Single-molecule surface-enhanced Raman spectroscopy (SM-SERS): characteristics and analysis