Metabolic　analysis　in　biofluids　based　on　laser　desorption/ionization　mass　spectrometry　(LDI-MS),　featuring　rapidity,　simplicity,　small　sample　volume　and　high　throughput,　is　expected　to　be　a　powerful　diagnostic　tool.　Nevertheless,　the　signals　of　most　metabolic　biomarkers　obtained　by　matrix-assisted　LDI-MS　are　too　limited　to　achieve　a　highly　accurate　diagnosis　due　to　serious　background　interference.　To　address　this　issue,　nanomaterials　have　been　frequently　adopted　in　LDI-MS　as　substrates.　However,　the　＇trial　and　error＇　approach　still　dominates　the　development　of　new　substrates.　Therefore,　rational　design　of　novel　LDI-MS　substrates　showing　high　desorption/ionization　efficiency　and　no　background　interference　is　extremely　desired.　Herein,　four　few-layered　organic　metal　chalcogenides　(OMCs)　were　precisely　designed　and　for　the　first　time　investigated　as　substrates　in　LDI-MS,　which　allowed　a　favorable　internal　energy　and　charge　transfer　by　changing　the　functional　groups　of　organic　ligands　and　metal　nodes.　As　a　result,　the　optimized　OMC-assisted　platform　satisfyingly　enhanced　the　mass　signal　by　≈10　000　fold　in　detecting　typical　metabolites　and　successfully　detected　different　saccharides.　In　addition,　a　high　accuracy　diagnosis　of　central　precocious　puberty　(CPP)　with　potential　biomarkers　of　12　metabolites　was　realized.　This　work　is　not　only　expected　to　provide　a　universal　detection　tool　for　large-scale　clinical　diagnosis,　but　also　provides　an　idea　for　the　design　and　selection　of　LDI-MS　substrates.　©　2024　The　Royal　Society　of　Chemistry.