A combined method for monitoring peptide coupling carrier protein prepared immunogen was established to get a carbodiimide coupling method which has the advantages of simple operation，high success rate and wide range of application. The optimized coupling method was applied in the preparation of immunogens for a variety of tumor-associated peptide biomarkers.Using synthesized SP0104 as the peptide，BSA （bovine serum albumin） as the carrier protein and EDC（carbodiimide） as the coupling reagent，the key conditions such as the activation method of reactants，the coupling time and the ratio of three reactants were investigated.The coupling ratio of UV and coupling rate of SDS-PAGE were used to monitor the compounds derived from the coupling reaction.The addition sequence of reagents was optimized as follows∶The peptide was mixed with BSA firstly，and then added into carbodiimide solution.The mass ratio of SP0104∶BSA∶EDC was 1∶1∶1, and coupling time was 12 h.The coupling ratio was between 4∶1 and 12∶1 and the coupling rate was between 9% and 20%.The antibody titer of purified SP0104 was 1∶80 000, and the polyclonal antibody successfully captured the peptide antigen SP0104 by the analysis of MALDI-TOF mass spectrometry.The optimized coupling method was applied in the peptide immunogen preparation of SPC09，SPC15，SPG01，SPG03 and SPG04.All the antibody titers of five immune-serum were over 1∶10 000，and the maximum reached up to 1∶512 000.The established combined monitoring method for carbodiimide coupling peptide and carrier protein was simple and effective，and could improve the success rate of peptide antibody preparation.