Fast and accurate pathogen detection in aquatic environments is challenging in many biomedical studies and microbial diagnostic applications. In this study, we developed a real-time, continuous, and non-destructive single cell detection method using target specific aptamer-conjugated fluorescent nanoparticles (A-FNPs) and an optofluidic particle-sensor platform. A-FNPs selectively bound to the surfaces of target bacteria ( Escherichia coli ) and labeled them with high affinity and selectivity so that target bacteria can be countable particles in an optofluidic particle-sensor. A-FNP-labeled target bacterial complexes were detected by the optofluidic particle-sensing system, which provides rapid and continuous single-cell detection. A-FNPs selectively bound to E. coli with a dissociation constant of 0.83 nM, but did not bind Enterobacter aerogenes or Citrobacter freundii strains, which lacked affinity for the aptamer used. We demonstrated that our optofluidic device achieves a detection throughput of ~100 particles per second with high accuracy (~85%) in detecting single bacterial cells conjugated with A-FNPs. This approach can be immediately extended to the real-time, high-throughput detection of other microorganisms such as viruses that are selectively conjugated with A-FNPs. Collectively, these data suggest that optofluidic systems are widely applicable for the fast and continuous detection of microbial cells. [ABSTRACT FROM AUTHOR]