This　work　presents　a　new　approach　for　the　analysis　of　small　molecules　with　direct　negative　ion　laser　desorption/ionization　(LDI)　on　graphene　flakes.　A　series　of　matrix　interference-free　mass　spectra　were　obtained　for　the　analysis　of　a　wide　range　of　small　molecules　including　peptides,　amino　acids,　fatty　acids,　as　well　as　nucleosides　and　nucleotides.　The　mixture　of　analytes　and　graphene　flakes　suspension　were　directly　pipetted,　onto　a　sample　plate　for　LDI-time-offlight　mass　spectrometry　(TOFMS)　analysis.　Deprotonated　monomeric　species　[M-H](+)　ions　were　homogeneously　obtained　on　uniform　graphene　flakes　film　when　negative　ion　mode　was　applied.　In　positive　ion　mode,　the　analytes　were　detected　in　form　of　multiple　adduct　ions　such　as　sodium　adduct　[M+Na](+),　potassium　adduct　[M+K](+),　double　sodium　adduct　[M+2Na-H](+)　double　potassium　adduct　[M+2K-H](+),　as　well　as　sodium　and　potassium　mixed　adduct　[M+Na+K-H](+).　Better　sensitivity　and　reproducibility　were　achieved　in　negative　ion　mode　compared　to　positive　ion　mode.　It　is　believed　that　the　new　method　of　matrix　interference　free　negative　ion　LDI　on　graphene　flakes　may　be　expanded　for　LDI-MS　analysis　of　various　small　molecules.