Although there have been many studies for the detection of Escherichia coli (E. coli), most methods still show limitations in that they are complex and time-consuming. To compensate for these shortcomings and to enable rapid and straightforward E. coli detection, we intend to utilize OmpT-a cell membrane endopeptidase of E. coli- as a suitable target molecule. OmpT, strongly embedded in the outer membrane of only E. coli and exposed to external solutions, exhibits high proteolytic activity. In this presentation, we would like to report a wash-free, sensitive, and selective amperometric method for E. coli detection, based on rapid and specific proteolytic cleavage by OmpT. The method involved (i) rapid proteolytic cleavage of consecutive amino acids linked to electrochemical species (4-aminophenol, AP) by leucine aminopeptidase (LAP, an exopeptidase) after initial cleavage by OmpT, (ii) affinity binding of E. coli on an electrode, and (iii) electrochemical-enzymatic (EN) redox cycling. OmpT cleaves the intermediate peptide bond of a peptide substrate containing alanine-arginine-arginine-leucine-AP (-A-R-R-L-AP) to form R-L-AP, followed by LAP sequentially cleaving the two peptide bonds of R-L-AP, liberating an electroactive AP. In addition to the rapid proteolytic cleavage by OmpT and LAP, affinity binding and EN redox cycling enabled high electrochemical signal amplification. This is the first time that two-step sequential cleavage has been introduced into protease-based detection, and the calculated detection limit for E. coli cells in tap water (approximately 103 CFU/mL after 1 h incubation) was lower than those obtained without affinity binding and EN redox cycling. The detection method was highly selective to E. coli as OmpT is present in only E. coli. High sensitivity, selectivity, and the absence of wash steps make the developed detection method practically promising.