Ultrathin　graphitic　carbon　nitride　(g-C3N4)　nanosheets　served　as　a　novel　matrix　for　the　detection　of　small　molecules　by　negative　ion　matrix-assisted　laser　desorption/ionization　time-of-flight　mass　spectrometry　(MALDI-TOF　MS)　was　described　for　the　first　time.　In　comparison　with　conventional　organic　matrices　and　graphene　matrix,　the　use　of　g-C3N4　nanosheet　matrix　showed　free　matrix　background　interference　and　increased　signal　intensity　in　the　analysis　of　amino　acids,　nucleobases,　peptides,　bisphenols　(BPs),　and　nitropolycyclic　aromatic　hydrocarbons　(nitro-PAHs).　A　systematic　comparison　of　g-C3N4　nanosheets　with　positive　and　negative　ion　modes　revealed　that　mass　spectra　produced　by　g-C3N4　nanosheets　in　negative　ion　mode　were　featured　by　singly　deprotonated　ion　without　matrix　interference,　which　was　rather　different　from　the　complicated　alkali　metal　complexes　in　positive　ion　mode.　Good　salt　tolerance　and　reproducibility　allowed　the　determination　of　1-nitropyrene　(1-NP)　in　sewage,　and　its　corresponding　detection　limit　was　lowered　to　1　pmol.　In　addition,　the　ionization　mechanism　of　the　g-C3N4　nanosheets　as　matrix　was　also　discussed.　The　work　expands　its　application　scope　of　g-C3N4　nanosheets　and　provides　an　alternative　approach　for　small　molecules.　©　2015　American　Chemical　Society.