Microfluidic lab-on-a-chip presents an ideal solution for bacterial sensing and identification due to its advantages like large surface-to-volume ratio, requirement of low sample volume and multiplexing possibility. The present work deals with the development of an immunosensor chip using circular microchannels fabricated directly with microdimensional copper wire and permanent magnet for capture of Fe3O4 magnetic nanoparticle (MNP) conjugate. The MNP facilitate capture of the antigen in a confined space and hence, enhanced fluorescence signal for detection. The multiplexed microfluidic chip permits visual detection and quantification of waterborne pathogens viz. Escherichia coli and Salmonella typhimurium simultaneously. CdTe quantum dots (QDs) with different emission wavelengths were conjugated with anti-E. coli and anti-S. typhimurium antibodies for concurrent fluorescence detection. The present technique provides an inexpensive yet powerful tool to image and quantify pathogens at low numbers with passage of large sample volumes.