A novel, fast and sensitive determination strategy for E. coli O157:H7 has been developed by combination of ligandmagnetic nanoparticles (LMNPs) enrichment with a fluorescent silica nanoparticles (FSiNPs) based two-color flow cytometry assay (LMNPs@FSiNPs-FCM). E. coli O157:H7 was first captured and enriched through the lectin concanavalin A (Con A) favored strong adhesion of E. coli O157:H7 to the mannose-conjugated magnetic nanoparticles. The enriched E. coli O157:H7 was further specially labeled with goat anti-E. coli O157:H7 antibody modified RuBpy-doped FSiNPs, and then stained with a nucleic acid dye SYBR Green I (SYBR-I). After dual-labeling with FSiNPs and SYBR-I, the enriched E. coli O157:H7 was determined using multiparameter FCM analysis. With this method, the detection sensitivity was greatly improved due to the LMNPs enrichment and the signal amplification of the FSiNPs labelling method. Furthermore, the false positives caused by aggregates of FSiNPs conjugates and nonspecific binding of FSiNPs to background debris could be significantly decreased. This assay allowed the detection of E. coli O157:H7 in PB buffer at levels as low as 7 cells mL(-1). The total assay time including E. coli O157:H7 sample enrichment and detection was less than 4 h. An artificially contaminated bottled mineral water sample with a concentration of 6 cells mL(-1) can be detected by this method. It is believed that the proposed method will find wide applications in biomedical fields demanding higher sensitive bacterial identification.