As a very promising spectral analytical technology, surface-enhanced Raman spectroscopy (SERS) has received extensive attention especially the applications in biomedical field. However, the SERS can only be obtained through the enhancement of SERS-active substrates, and further development of SERS is mainly limited by the reproducible preparation of clean and highly surface enhanced Raman scattering (SERS) active substrates. The application of nanotechnology during preparing SERS-active substrates must be one of the best solutions. In this paper, various methods will be introduced for preparing SERS-active substrates. Unfortunately, at present, it is still difficult to obtain SERS substrates that can simultaneously meet quantitative and trace analysis requirements by one method. Emphasis of this paper is placed on the analysis of the advantages and weaknesses of different methods in preparing SERS-active substrates. Well-dispersed nanoparticles and highly ordered substrates can be used for quantitative analysis while the nanoparticle aggregates with extremely high sensitivity can be effectively used for qualitative analysis. So, proper fabrication methods should be selected for a special purpose of SERS.