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Rapid, quantitative, and sensitive assays for the multiplexed detection of bacterial pathogens are urgently needed for public health. Here, we report the generation of dual-modified phage sensors for the simultaneous detection of multiple pathogenic bacteria. The M13KE phage was dual modified to display the targeting peptide on the minor coat protein pIII (∼5 copies) and the streptavidin-binding (StrB) peptide on the major coat protein pVIII (∼2700 copies). The targeting peptide specifically recognizes the target bacteria, and the StrB peptide acts as the efficient signal amplification and transduction unit upon binding with fluorescently tagged streptavidin. The bright fluorescence emitted from individual target bacteria can be clearly distinguished from the background via both the flow cytometry and fluorescence microscopy. Three different dual-modified phages targeting E. coli O157:H7, Salmonella Typhimurium, and Pseudomonas aeruginosa were constructed, and high specificity was verified via a large excess of other non-target bacteria. Using a 40 mL sample volume, the target bacteria detection limit was approximately 102 cells/mL via flow cytometry measurement in the presence of other non-target bacteria. By combining these three dual-modified phages into a cocktail, simultaneous detection and quantification of three target bacterial pathogens was demonstrated with good linearity. The strategy of constructing dual-modified phage represents a promising tool in the detection of bacterial pathogens. [Display omitted] • Using dual-modified phage, a rapid and sensitive method was developed for the specific detection of bacterial pathogens. • Phages targeting E. coli O157:H7, S. Typhimurium, and P. aeruginosa were constructed and verified with high specificity. • The bright fluorescence of single target bacteria can be readily detected by flow cytometry and fluorescence microscopy. • The detection limit of the target bacteria was approximately 102 cells/mL via flow cytometric measurement. • By using phage cocktail, simultaneous detection and linearity quantification of three target bacterial pathogens was demonstrated. [ABSTRACT FROM AUTHOR]