Staphylococcal　enterotoxin　A　(SEA)　was　the　major　virulence　factor　of　Staphylococcus　aureus　and　a　biomarker　of　S.　aureus.　To　establish　a　fast,　low　cost,　high　accuracy,　reliable,　and　simple　method　for　detecting　S.　aureus,　SEA　was　analyzed　by　HPLC-ESI-TOF.　SEA　was　not　yet　commercially　available　in　universal,　so　SEA　was　prepared　before　it　was　analyzed　by　HPLC-ESI-TOF.　The　result　showed　that　high　purified　SEA　was　successfully　prepared　and　SEA　has　normal　distribution　in　mass　spectra.　A　large　amount　of　recombinant　SEA　(rSEA)　was　obtained　by　engineering　technology　and　was　purified　by　Ni　affinity　chromatography　column,　and　the　expression　and　purity　of　rSEA　and　SEA　were　analyzed　by　SDS-PAGE.　The　factors　effected　on　ionization　of　SEA　were　studied,　and　the　qualitative　analysis　of　SEA　by　HPLC-ESI-TOF.　The　result　showed　that　large　amount　of　SEs　expressed　within　a　short　time　at　28　A　degrees　C　or　thereabouts,　and　there　was　no　impurity　bands　in　electrophorogram　after　rSEA　was　purified　by　Ni　affinity　chromatography　column.　In　addition,　the　SEA　which　had　homologous　AA　sequence　with　wild　SEA　was　made　by　rSEA.　The　retention　of　SEA　in　column　and　ionization　of　SEA　in　ESI-TOF　were　studied　for　qualitative　analysis　of　S.　aureus.　The　result　showed　that　the　content　of　formic　acid　in　mobile　phase　was　an　important　factor　for　ionization　of　SEs　in　ESI-TOF.　And　the　result　provided　theoretical　foundation　for　qualitative　detection　of　S.　aureus.　[SEs　+　nH(+)　+　mNH(4)　(+)]　(n+m+)　was　shown　on　ESI-TOF　spectra　when　SEA　was　detected　by　ESI-TOF　in　positive　ion　mode,　and　the　numerical　value　of　n+m　was　less　than　or　equal　to　the　number　of　basic　amino　acids　in　SEs.　This　method　was　applied　to　determine　SEA　in　water　samples　preliminarily,　and　the　detection　limit　of　SEA　in　spiked　water　sample　was　3　mg/kg.　The　limit　of　detection　of　3　mg/kg　was　low　sensitivity　for　low　molecular　weight　matters,　but　it　was　high　sensitivity　for　SEA　which　had　a　high　molecular　weight　of　27　kDa.　Of　SEA,　3　mg/kg　was　equivalent　to　10(-4)　mmol/kg　of　SEA.　This　study　can　provide　evidence　for　establishing　method　to　determine　SEA　in　real　samples.