Because the hydrophobic surface can reduce the diffusion of probe molecules and enrich the detection materials effectively, two types of hydrophobic Surface-enhanced Raman spectroscopy (SERS) substrates were prepared in this work. Cotton fabrics were used as raw material, employing printing and spraying methods to fabricate substrates with various levels of hydrophobicity. A silver layer was deposited on the surface of the hydrophobic substrates by vacuum evaporation to prepare hydrophobic and super-hydrophobic SERS substrates, and p-aminothiophenol (PATP) was used as probe molecule to explore SERS performance of different samples. The analyzed results show that the enhancement factor of the optimized hydrophobic SERS substrate is 1.57 × 106, and the detection sensitivity can reach up to 10−10 M and the relative standard deviation (RSD) value is 11.19%. The enhancement factor of the optimized super-hydrophobic SERS substrate is 1.93 × 106, its detection limit is low to 10−11 M, RSD value is 8.11%, and it still has the effect of enhancing Raman signal after 6 months of storage. The detection limit of the optimized super-hydrophobic substrate for lead ion solution is as low as 200 nM. Compared with hydrophilic substrates, the SERS performances of the substrates with hydrophobic surface are improved significantly.