Isoelectric focusing in thin-layer polyacrylamide gels(IEF-PAGE) were applied to analyze lipoxygenase lacking types near iosogenic line(NIL) of soybeans.Seeds with marked types were managed by degreasing,centrifugating and drying with vacuumized.Three lipoxygenase isozymes were separated clearly from crude seed extracts by native polyacrylamide gel eletrophoretic technique,and three protein bands were eluted and purified right.Considered activity and purity of three lipoxygenases,Lipoxygenase-1 can be regarded as appropriate antigen.The minimum amount of antiserum could successfully detect 10 ug/ml antigen with 1:1600 dilution of antiserum by indirect enzyme-linked immunosorbent assay(ELISA).Hybridoma cell lines of secreting monoclonal antibodies(MAbs) were fused with myeloma cells(SP2/0) and spleen cells from BALB/C mouse,which immunized by Lox1 particles.Three MAbs present positive through limited diluting and cross-reacting by Indirect ELISA,and do not cross-react with other tested soybean lipoxygenase proteins.Then,monoclonal antibodies using hybridoma fusion can identify isozyme to be present or absent,and this test results can provide an efficient and cheap determining method to assist breeding and experiment base in order to develop reagent box.