CuFe2O4　magnetic　nanocrystal　clusters　(CuFe2O4　MNCs)　were　proposed　as　a　new　matrix　for　small　molecule　analysis　by　negative　ion　matrix-assisted　laser　desorption/ionization　time-of-flight　mass　spectrometry　(MALDI-TOF　MS)　for　the　first　time.　We　demonstrated　its　advantages　over　conventional　organic　matrices　in　the　detection　of　small　molecules　such　as　amino　acids,　peptides,　nucleobases,　fatty　acids,　and　steroid　hormones.　A　systematic　comparison　of　CuFe2O4　MNCs　with　different　ionization　modes　revealed　that　MS　spectra　obtained　for　the　CuFe2O4　MNC　matrix　in　the　negative　ion　mode　was　only　featured　by　deprotonated　ion　peaks　with　a　free　matrix　background,　which　was　different　from　the　complicated　alkali　metal　adducts　produced　in　the　positive　ion　mode.　The　developed　method　was　found　relatively　tolerant　to　salt　contamination　and　exhibited　good　reproducibility.　A　detection　limit　down　to　the　subpicomolar　level　was　achieved　when　testosterone　was　analyzed.　In　addition,　by　comparison　of　the　MS　spectra　obtained　from　bare　Fe3O4　and　MFe2O4　MNC　(M　=　Co,　Ni,　Cu,　Zn)　matrices,　two　main　factors　of　MFe2O4　MNC　matrices　were　revealed　to　play　a　vital　role　in　assisting　the　negative　ion　desorption/ionization　(D/I)　process:　doping　transition　metals　into　ferrite　nanocrystals　favoring　laser　absorption　and　energy　transfer　and　a　good　match　between　the　UV　absorption　of　MFe2O4　MNCs　and　the　excitation　of　nitrogen　laser　source　facilitating　LDI　efficiency.　This　work　creates　a　new　branch　of　application　for　MFe2O4　MNCs　and　provides　an　alternative　solution　for　small　molecule　analysis.