We　report　a　three-dimensional　(3D)　SERS　substrate　consisting　of　a　silver　nanoparticle　(AgNP)　coating　on　the　skeleton-fiber　surfaces　of　a　polytetrafluoroethylene　(PTFE)　membrane.　Simple　thermal　evaporation　was　employed　to　deposit　Ag　onto　the　PTFE　membrane　to　produce　grape-shaped　AgNPs.　The　3D-distributed　AgNPs　exhibit　not　only　strong　localized　surface　plasmon　resonance　(LSPR)　but　also　strong　hydrophobic　performance.　High-density　hotspots　via　silver　nano-grape　structures　and　nanogaps,　the　large　3D　interaction　volume,　and　the　large　total　surface　area,　in　combination　with　the　hydrophobic　enrichment　of　the　specimen,　facilitate　high-sensitivity　sensing　performance　of　such　a　SERS　substrate　for　the　direct　detection　of　low-concentration　molecules　in　water.　An　enhancement　factor　of　up　to　1.97　×　1010　was　achieved　in　the　direct　detection　of　R6G　molecules　in　water　with　a　concentration　of　10−13　mol/L.　The　lowest　detection　limit　of　100　ppt　was　reached　in　the　detection　of　melamine　in　water.　Such　a　SERS　sensor　may　have　potential　applications　in　food-safety　control,　environmental　water　pollution　monitoring,　and　biomedical　analysis.　©　2024　by　the　authors.