In single molecule study, surface-enhanced Raman scattering (SERS) has the advantage of specifically providing structural information of the molecules targeted. The main challenge in single molecule SERS is developing reusable plasmonic substrates that ensures single molecule sensitivity and acquires intrinsic information of molecules. Here, we proposed a strategy to utilize single- walled carbon nanotubes (SWNTs) to construct SERS substrates. Employing ultrasonic spray pyrolysis, we prepared in situ polyhedral gold nanocrystals closely spaced and attached to nanotubes, ensuring valid hot spots formed along the tube-walls. With such SERS substrates, we proved the single molecule detection by the statistical analysis based on the natural abundance of isotopes. Since SWNTs provide non-chemical bonding adsorption sites, our SERS substrates are easily reusable and have a unique advantage of preserving the intrinsic property of the molecules detected. Using SWNTs to build SERS substrates may become a powerful general strategy in various static and dynamic studies of single molecules.