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A rapid and highly sensitive chemiluminescence biosensor combined with a microfluidic filter chip was investigated and evaluated for the detection of E. coli O157:H7. The microfluidic filter chip consisted of a microchamber and a microchannel glass chips bonded by thermal fusion bonding to form a stepped channel configuration. The filter chip was designed with multiple outlets. The dimension of the microchamber was 1 mm x 1 mm x 11.5 m. Microchamber was used for the formation of single layer of magnetic beads. The sample containing E. coli O157:H7 was mixed with immunomagnetic microbeads and peroxidase labeled anti-E. coli O157:H7 antibodies to form sandwich complexes. A syringe pump was used to inject the sandwich complexes into the filter chip, and luminol was added to generate a chemiluminescence signal, which was recorded in a real time with a fiber optic light guide connected to a luminometer coupled to data acquisition unit and PC. The result indicated that this filter chip could be used for the detection of as low as 800 CFU/ml of E. coli O157:H7 without enrichment. A multiple enrichment sampling methodology was used for the higher capture of E. coli O157:H7 by the microbeads. We demonstrated that new sampling technique maximized the capture efficiency of magnetic beads and a detection limit of 180 CFU/ml was achieved with an improved sampling technique. The total time of detection was 90 min. Thus, highly sensitive chemiluminescence biosensor combined with the microfluidic filter chip has potential to detect very low cell number of target pathogenic bacteria.