Abstract: Objective To identify the bacteria after multiple passages by the application of peptide mass fingerprinting technology, and analyze the influence of bacterial culture on the ability of bacterial identification. Methods The strains of Helicobacter pylori, Escherichia coli and Staphylococcus aureus were continuously cultured for 57 generations, 50 generations and 100 generations respectively, and antigen of S. aureus was collected.Each generation of fresh cultured bacteria and frozen antigen protein were extractedby ethanol/formic acid method.The peptide mass fingerprinting technique was used for the spectrum acquisition and identification of the strains.NanoLC-MS/MS were used to reveal the protein components of H. pylori. Results H. pylori and E. coli were continuously cultured for 57 generations and 50 generations respectively, every generation was identified at the species level by the peptide mass fingerprint spectrum. S. aureus was continuously cultured for 100 generations, fresh strain and frozen antigen were identified at the species level by the peptide mass fingerprint spectrum.The peptide mass fingerprint spectra of each generation of frozen and fresh strains had good consistency.A total of 206 proteins of H. pylori were identified through protein scanning analysis, including 29.6% of enzymes, 15.5% of ribosomal protein, 10.7% of out membrane protein, 19.0% of putative protein, 2.0% of transport relative protein and 22.0% of the others. Conclusion Continuous passage of bacteria and frozen antigen have no affects on the correct identification of peptide mass fingerprint spectrum, and the quality of the peptide mass fingerprint is stable.