Abstract: Objective：To identify E.coli from positive blood cultures rapidly by the surface enhanced laser desorption & ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Methods：Firstly, the protein fingerprint model of E.coli was constructed by analyzing 19 strains of E.coli with the SELDI-TOF-MS. Then, 168 isolates from clinical samples were detected with the SELDI-TOF-MS, and the obtained data were compared with the protein fingerprint for evaluating the model. Finally, 88 cases of positive blood cultures were analyzed with the SELDI-TOF-MS and the obtained data were preformed the resemblance comparison to the protein fingerprint for identifying the infection of E.coli. Results：The protein fingerprint model of E.coli was successfully constructed from 19 strains of E.coli, which were demonstrated by PCR and DNA sequencing, and the coincidence rate was 100% for 168 isolates. The identification for 88 cases of positive blood cultures showed that there was high concordance (k=0.905) between the SELDI-TOF-MS and the conventional pathogen cultures. Conclusion：The SELDI-TOF-MS could identify E.coli from positive blood cultures rapidly, which provided a new approach for the rapid diagnosis of blood infection.