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Rapid and accurate bioburden detection has become increasingly necessary for food, health, pharmaceutical and environmental applications. To detect bioburden accurately, and in a highly sensitive manner, we have fabricated a novel microfluidic device with an integrated filter to trap the cells. Bioburden is detected on the filter paper in situ using the redox reaction of fluorescent label resorufin and a portable multichannel fluorometer is used for fluorescence measurement. The microfluidic device was fabricated in a facile, low-cost, and rapid way with microwave-induced thermally assisted bonding. To characterize the bonding quality of the microfluidic cassettes, different tests were performed, and the filter paper material and size were optimized. Primary Bacillus subtilis culture bacterial samples were filtered through the device to validate and investigate the performance parameters. Our results show that a limit of detection (LOD) of 0.037 CFU/mL can be achieved through this microfluidic device whereas the LOD in a normal microfluidic cassette in the fluorometer and the golden standard spectrophotometer are 0.378 and 0.128 CFU/mL respectively. The results depict that three to ten times LOD improvement is possible through this microfluidic cassette and more sensitive detection is possible depending on the volume filtered within a rapid 3 min. This novel microfluidic device along with the fluorometer can be used as a rapid portable tool for highly sensitive, accurate and high-throughput bacterial detection for different applications. [ABSTRACT FROM AUTHOR]