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Herein, we present a microfluidic-based point-of-care testing approach for viable Escherichia coli detection in tap water. In this approach, antibody-modified magnetic particles were used for capturing E. coli from water, followed by concentrating the bacteria-bound particles in the device using an external magnet. Then, redox indicator reagent mixed culture medium was added to the device, which allows colorimetric signal change as a result of reduction reaction linked to bacteria proliferation. Corresponding color change was monitored from microfluidic access holes, which is named μ-pupil, in which the colorimetric signal can be recorded from two perspective view angles using a portable mobile-phone platform, and analyzed using MATLAB software. The correlation of the colorimetric signal and E. coli concentration showed linearity within the range of 8ˣ10 3–8× 10 5 CFU/ml E. coli and 8× 10 0–8× 10 3 CFU/ml E. coli after 6 h and 12 h of incubation, respectively. The small size of the microfluidic device is found advantageous for shortening the viable detection time from 21 hours to 12 hours. The transparency of the device allows to monitor the colorimetric signal change even with a naked eye, while shallow microfluidic channel with the depth of 14 μm and glass coverslip bottom layer facilitates fluorescence signal measurement for probing chlorine stress effects on viable bacteria. The developed μ-pupil device-based point-of-care testing approach, which allows low concentrations of viable E. coli detection in less than 13 hours of total detection time, is promising for future point-of-care-testing of fecal indicator E. coli in water.