With the rapid development of human's exploitation of nature and animal husbandry, zoonoses have become a major public health problem worldwide. It is necessary to establish a rapid, specific and sensitive detection method to screen several zoonotic pathogenic bacteria simultaneously. In this study, phage display technology was used to screen specific peptide of three common zoonotic pathogens, E. coli O157:H7, L. monocytogenes and B. melitensis 16 M. Then, three peptide were obtained, named E2, L4 and B4, which can identify the three pathogens respectively. Three peptide modified with biotin were synthesized and were coupled to streptavidin magnetic beads (MBs) to form peptide-MBs, which enriched the above three pathogens from the samples. Three quantum dot (QD) probes were constructed by coupling three polyclonal antibodies to different fluorescent QD surfaces (QD540, QD580 and QD630). The simultaneous detection method based on peptide-MBs and QDs multicolor fluorescent labeling was established and could detect E. coli O157:H7, L. monocytogenes and B. melitensis 16 M simultaneously. The detection method took about 100 min with the detection limits of 103, 102 and 102 CFU/mL, respectively. The detection method could be also well utilized in real samples. Display omitted •Three peptides which could recognize E. coli O157:H7, L. monocytogenes and B. melitensis 16 M were screened successfully by phage display technology, with the advantages of good specificity, clear structure and low cost.•Peptide-MBs were utilized to enrich and separate pathogens in samples quickly and conveniently.•Multicolor fluorescent labeling of QDs have been successfully used for simultaneous detection of various pathogens.•The simultaneous fluorescence detection method was satisfactorily applied to the detection of zoonosis pathogens.