This paper first briefly introduces the Raman signal enhancement principle of surface enhanced Raman scattering (SERS), the research and application progress of the conventional SERS substrate is presented, then the definition and advantages of hydrophobic substrates are described along with the classification of SERS substrate with progress in research and application. The development trend and direction of hydrophobic SERS substrates are outlined as well. Fabrication and application of hydrophobic SERS substrates will provide sensitive and reliable methods and new ideas for the detection of ultra-low concentration of biochemical substances and detection and diagnosis of diseases based on human bodily fluids.