The　chemical　enhancement　of　semiconductor-based　surface-enhanced　Raman　scattering　(SERS)　substrates　is　an　exciting　hot　topic.　Herein,　a　simple　hydrothermal　method　is　developed　to　prepare　molybdenum　disulfide　(MoS2),　which　can　be　easily　exfoliated　into　monolayer　nanosheets　(MoS2NSs)　by　sonication,　even　in　the　absence　of　any　surfactant.　The　obtained　MoS2NSs　contain　two　types　of　defects,　namely,　one　caused　by　the　incorporation　of　Mo　atoms　of　high　valence　states　and　one　caused　by　the　incorporation　of　S22-.　The　density　of　the　two　types　of　defects　can　be　easily　tuned　by　controlling　the　ratio　of　Na2S　and　Na2MoO4　in　the　raw　materials.　The　unique　properties　and　the　clear　surface　make　the　obtained　MoS2NSs　ideal　models　to　investigate　the　effect　of　defects　on　the　SERS　activity　of　MoS2.　It　is　found　that　the　SERS　activity　of　the　obtained　MoS2NSs　increases　dramatically　with　the　defects　caused　by　Mo　atoms　of　high　valence　states,　while　it　first　increases　and　then　decreases　with　the　increase　of　defects　caused　by　S22-.　On　the　basis,　MoS2NSs　with　high　SERS　activity　and　a　low　detection　limit　of　5.0　×　10-9　mol/L　toward　crystal　violet　(CV)　are　obtained.　Moreover,　the　mechanism　of　defects　affecting　the　SERS　activity　of　MoS2NSs　is　revealed.　The　defects　on　one　hand　provide　a　large　amount　of　dangling　bonds　that　can　combine　CV　molecules　to　form　MoS2NS-CV　complex　and　on　the　other　hand　provide　extensive　induced　local　dipoles　and　enhance　the　overall　SERS　spectrum　of　CV.　©　2024　American　Chemical　Society.