Sensitive and rapid detection of E. coli is important for food safety. Existing methods are limited in detection time or discriminating viable/dead bacteria. In this work, we report a rapid and sensitive method based on M13K07 phage whose pVIII proteins (about 2700 copies) are biomodified by streptavidin binding peptides (SBP)-tag for live E. coli analysis. The M13K07-SBP was used to inoculate contaminated food. After specific infection and rapid propagation of M13K07-SBP in live E. coli, plentiful progeny phages carried SBP tags on pVIII proteins that can load numerous signal transducers were yielded for signal amplification output. Owing to the replication and the great loading capacity of M13K07-SBP, as low as 4 cfu/mL of E. coli was detected in less than 3.5 h. Moreover, specific analysis and practical application were successfully confirmed. In brief, this work shows great potential in providing sensitive, rapid, and specific detection method for viable E. coli.