Although immunomagnetic separation is a useful sample pretreatment method that can be used to separate target pathogens from a raw sample, it is challenging to remove unbound free magnetic nanoparticles (MNPs) for colorimetric detection of target pathogens. Here, size-based filtration was exploited for the rapid on-site detection of pathogens separated by immunomagnetic separation in order to remove unbound free MNPs using a finger-powered microfluidic device. A membrane filter and an absorbent pad were integrated into the device and a mixture of unbound free MNPs and MNP-bound Escherichia coli (E. coli) O157:H7 was dispensed over the membrane filter by pressing and releasing the pressure chamber. A colorimetric signal was generated by MNP-bound E. coli O157:H7 while unbound free MNPs were washed out by the absorbent. Furthermore, the colorimetric signals can be amplified using a gold enhancer solution when gold-coated MNPs were used instead of MNPs. As a result, 10(2) CFU/mL E. coli O157:H7 could be detected by the enhanced colorimetric signal on a proposed device.