We report the development of automated flourometric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating fluorophore as an analytical reagent. Thiolated antibodies (anti-E. coli O157:H7) were coupled to malemide-tagged liposomes encapsulating dye. To automate the assay, a fluorescence plate reader was included in the assay system to detect fluorophore released from lysed liposomes in a microplate. The detection limit of the current assay with pure cultures of the serotype was about 104 colony-forming units (CFU)/mL. The assay can detect E. coli O157 in ground beef samples inoculated with as few as 0.8 CFU/mL after a 12-h enrichment. These results demonstrate the feasibility of using fluorophore-encapsulated immunoliposomes in a microtiter plate for the rapid and automated detection of molecules with multivalent antigenic sites.