<jats:title>Abstract</jats:title> <jats:p>Infectious disease mortality has decreased due to effective drugs and healthcare. However, global health remains threatened by infectious diseases. New methods of rapid and accurate bacterial detection have attracted considerable attention. Fluorescence detection of whole bacterial cells offers high sensitivity, quantitative analysis, and simple operation. A highly fluorescent bioconjugated probe improves sensitivity and selectivity. This study presents a novel, bright fluorescent probe comprising a bacteriophage and a fluorescent nanoemulsion (fNE) as biorecognition and signal transduction elements, respectively. We demonstrate that fluorescence microscopy imaging using the S. aureus-specific phage, S13′-fNE (phage-fNE), detects S. aureus in the presence of E. coli or S. pseudintermedius, another closely related Staphylococci, in a highly selective manner. Furthermore, fNEs with high dye loadings exhibit considerably greater brightness compared to the fluorescent dye alone, making them suitable for sensitive fluorescence imaging. Phage-fNEs can quantitatively detect S. aureus at 104–108 colony-forming units per milliliter (CFU mL−1), with a limit of detection of 8 × 104 CFU mL−1. This result is comparable to the lowest value achieved by microscopic bacterial detection, with no preconcentration or enzymatic signal enhancement methods used. Bioconjugated fNEs open new avenues for highly selective and sensitive fluorescent detection of bacteria.</jats:p>