A novel electrogenerated chemiluminescence (ECL) biosensor for highly sensitive detection of Escherichia coli (E. coli) was first developed by employing Concanavalin A (Con A) as a biological recognition element and bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine ruthenium (II) (Ru1) complex as the detector. The ECL biosensor was fabricated by adsorbing carboxyl-functionalised single-wall carbon nanotubes (SWNTs) onto a paraffin-impregnated graphite electrode and further covalently coupling the Ru1–Con A probe onto the surface of the SWNT-modified electrode. Upon the binding of E. coli O157:H7 (as a model target), the biosensor showed a decreased ECL intensity in the presence of tri-n-propylamine (TPrA), which was in logarithmically direct proportion to the concentration of E. coli over a range from 5.0×102 to 5.0×105cells/mL. The detection limit of this sensor was 127cells/mL. Additionally, the ECL biosensor also showed satisfactory selectivity in discriminating gram-negative E. coli from gram-positive bacteria. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and rapid detection of other desired bacteria.