Multiplex detection and quantification of bacteria in water by using portable devices are particularly essential in low and middle-income countries where access to clean drinking water is limited. Addressing this crucial problem, we report a highly sensitive immunoassay sensor system utilizing the fluorescence technique with magnetic nanoparticles (MNPs) to separate target bacteria and two different types of quantum dots (CdTe and Ni doped CdTe QDs) incorporated into a passive microfluidic chip to transport and to form sandwich complexes for the detection of two target bacteria, namelyEscherichia coli(E. coli) andSalmonella enteritidis(S. enteritidis) in less than 60 min. The assay is carried out on a capillary driven microfluidic chip that can be operated by merely pipetting the samples and reagents, and fluorescence measurements are done by using a handheld fluorescence spectrophotometer, which renders the system portable. The linear range of the method was found to be 10(1)to 10(5)cfu mL(-1)for bothE. coliandS. enteritidis. The limit of detection (LOD) was calculated to be 5 and 3 cfu mL(-1)forE. coliandS. enteritidis, respectively. The selectivity of the method was examined by testingEnterobacter dissolvens(E. dissolvens) andStaphylococcus aureus(S. aureus) samples, and no significant interference was observed. The method was also demonstrated to detect bacteria in tap water and lake water samples spiked with target bacteria.