Glassy　carbon　(GC)　as　a　well-known　electrode　material　has　recently　been　proposed　to　consist　of　fullerene-like　nanostructures.　In　order　to　verify　the　nanostructures　in　GC,　find　more　physiochemical　properties　of　GC,　and　develop　sensors　based　on　GC-related　carbon　nanomaterials,　we　investigated　the　morphologies　and　surface　states　of　GC　microspheres　(GCMs)　and　their　HNO3-oxidized　products　(ox-GCMs)　with　scanning　electron　microscopy　(SEM),　electrochemiluminescence　(ECL),　Raman　spectroscopy,　X-ray　photoelectron　spectroscopy　(XPS),　and　electron-paramagnetic　resonance　(EPR)　spectroscopy.　Our　research　results　reveal　that　ox-GCMs　rather　than　raw　GCMs　have　abundant　surface　states,　including　many　carboxyl　groups　(-COOH),　surface　defects　(or　carbon　edges),　and　C-related　dandling　bonds.　The　surface　states　with　a　band　gap　of　2.14　eV　endow　ox-GCMs　with　strong　cathodic　ECL　activity　in　the　presence　of　peroxydisulfate　(S2O82-).　The　ECL　behaviors　and　maximum　emission　wavelength　(580　nm)　of　ox-GCMs　are　very　similar　to　those　of　small-sized　graphene　quantum　dots　and　fullerene-like　nanosheets,　verifying　that　GCMs　are　essentially　3-D　nanomaterials　consisting　of　graphene　or　fullerene-like　carbon　nanostructures.　It　is　for　the　first　time　that　a　microsized　carbon　material　was　reported　to　have　good　ECL　activity　in　aqueous　media.　Possible　mechanisms　for　surface　state　formation　and　ECL　reactions　are　proposed　for　ox-GCMs,　and　a　promising　application　of　ox-GCMs　in　ECL　immunosensing　has　been　demonstrated　by　determining　prostate　specific　antigen　(PSA)　as　a　model　cancer　biomarker.　Copyright　©　2020　American　Chemical　Society.